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  • Articles  (640)
  • Life Sciences  (640)
  • 1975-1979  (515)
  • 1970-1974  (125)
  • 1
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 79-96 
    ISSN: 0091-7419
    Keywords: red cell membrane proteins ; spectrin ; red cell shape ; deformability ; membrane protein cross-linking ; membrane protein disulfide coupling ; red cell adenosine triphosphate ; calcium ; membrane protein polymerization ; discocyte-echinocyte transformation ; irreversibly sickled cells ; sickle cell anemia ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It has been proposed that the spectrin-actin submembrane network participates in control of red cell shape and deformability. We have examined ATP- and calcium-dependent changes in organization of spectrin in the membrane employing cross-linking of the nearest membrane protein neighbors by spontaneous or catalyzed (CuSO4, O-phenanthroline) intermolecular disulfide couplings and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis.Cross-linking of fresh red cells resulted in the formation of spectrin and actin dimers and tetramers. ATP-depleted red cells differed from fresh cells in the presence of an additional reducible polymer of MW 〉 1 × 106 selectively enriched in spectrin. This polymer formed spontaneously when red cells were depleted of ATP under aerobic conditions. After anaerobic ATP depletion, the polymer formed in ghosts after cross-linking by catalytic oxidation. Polymerization was prevented by maintenance of ATP and coincided with an ATP-dependent discocyte-echinocyte transformation. This suggests that, in ATP-depleted red cells, spectrin is rearranged to establish closer contacts, and that this may contribute to the discocyte-echinocyte transformation.The introduction of greater than 0.5 mM Ca++ into ghosts by inclusion in hemolysis buffer or into fresh red cells (but not ATP-depleted red cells) by treatment with ionophore A23187 spontaneously produced a nonreducible polymer which others have attributed to transamidative cross-linking of spectrin, band 3, and other proteins. Spontaneous formation of both polymer types (reducible in aerobically ATP-depleted red cells and nonreducible in fresh, Ca++ enriched red cells) resulted in stabilization (“autocatalytic fixation”) of spheroechinocytic shape.Irreversibly sickled cells, which have increased calcium and decreased ATP, and exhibit a permanent membrane deformation, failed to form any of the above polymers. This suggests that in contrast to normal cells depleted of ATP in vitro, fixation of ISC shape in vivo is not related to Ca- and ATP-dependent membrane protein polymerization. However, ISCs had an increased propensity to form the reducible, spectrin-rich polymer during a subsequent metabolic depletion in vitro. This was associated with transformation of ISCs into spheroechinocytes. Similar echinocytic ISCs were found to constitute 5-10% of the densest fractions of freshly separated ISCs. ISCs then exhibit sphero-echniocyte transformation, both in vitro and in vivo. We propose that this is due to spectrin reorganization that presumably results from the progressively increasing calcium and decreasing ATP of ISCs.These data provide evidence of altered spectrin organization in membranes of ATP-depleted, calcium-enriched red cells in vitro and in vivo.
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  • 2
    ISSN: 0091-7419
    Keywords: virus assembly ; limited proteolysis ; conformational change ; cooperativity ; electron microscopy ; optical diffraction ; computer image processing ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Giant T4 phage capsoids formed in canavanine-treated cultures infected by phage mutants in genes 21 and 17, respectively, differ with regard to cleavage of the major capsid protein, gp 23, and in the fine structure of their hexagonal surface lattices. Quantitative computer processing of electron micrographs shows that the significant differences in capsomer morphology amount to six symmetrically placed features present in the uncleaved hexamer but absent after cleavage. These features may be related with the N-terminal portions of gp 23 monomers excised by phage-specific proteolysis. Cleaved 17- giants can be induced to undergo a further structural transformation (expansion). Structural characteristics of partially transformed giant particles give clues about the dynamics of the cleavage and expansion transformations. Both processes appear to be polar, initiating in one cap and propagating along the particle. The transition zone of partial cleavage is diffuse, whereas the transition between unexpanded and expanded areas is confined to a narrow band of some 20 nm width.
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  • 3
    ISSN: 0091-7419
    Keywords: vertebrate photoreceptor ; cyclic GMP ; cyclic nucleotide regulation ; phosphodiesterase ; light activated GTPase ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3′5′-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light-dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.
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  • 4
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of 3H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6-8 h period as an obligatory step in production of “second messenger” in the action of this hormone.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 227-239 
    ISSN: 0091-7419
    Keywords: spectrin ; actin ; red cell membranes ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The human erythrocyte structural protein spectrin and its subunits I, II were isolated in the presence of Na-dodecyl-sulfate by gel filtration and preparative gel electrophoresis. After removal of the detergent, spectrin alpha-helical content is comparable to spectrin isolated without detergent. Subunits I and II formed single bands in isoelectric focusing (pI = 5.6) and in Ornstein-Davis disc gel electrophoresis systems, indicating the individual subunits are homogenous in nature. The molecular weights of the subunits I and II, determined by Ferguson plot, are 237,500 and 238,600, respectively, which is in good agreement with values obtained by the standard SDS gel relative mobility method. Limited tryptic digestion of spectrin and two-dimensional peptide maps of the individual subunits cleaved by S-cyanylation reaction showed dissimilar patterns, suggesting differences in primary structure between the two subunits.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 7
    ISSN: 0091-7419
    Keywords: membrane hydration ; membrane-bound water ; ANS fluorescence ; infrared spectra ; water-membrane interactions ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bound water is a major component of biological membranes and is required for the structural stability of the lipid bilayer. It has also been postulated that it is involved in water transport, membrane fusion, and mobility of membrane proteins and lipids. We have measured the fluorescence emission of membrane-bound 1-anilino-8-naphthalenesulfonate (ANS) and the infrared spectra of membranes, both as a function of hydration. ANS fluorescence is sensitive to polarity and fluidity of the membrane-aqueous interface, while infrared absorption is sensitive to the hydrogen bonding and vibrational motion of water and membrane proteins and lipids. The fluorescence results provide evidence of increasing rigidity and/or decreasing polarity of the membrane-aqueous interface with removal of water. The membrane infrared spectra show prominent hydration-dependent changes in a number of bands with possible assignments to cholesterol (vinyl CH bend, OH stretch), protein (amide A, II, V), and bound water (OH stretch). Further characterization of the bound water should allow its incorporation into current models of membrane structure and give insight into the role of membrane hydration in cell surface function.
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  • 8
    ISSN: 0091-7419
    Keywords: antigenic membrane glycoproteins ; immunoprecipitation ; two-dimensional electrophoresis ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (3H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (125I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated Mr̄ and pI. Of the 33 3H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with 125I, confirming that these are glycoproteins.The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, Mr̄, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.
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  • 9
    ISSN: 0091-7419
    Keywords: fibroblasts ; plasma membranes ; contact inhibition ; growth control ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G1 protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 11
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 359-364 
    ISSN: 0091-7419
    Keywords: ribosomes ; crystallization ; hypothermia ; chick embryos ; degeneration ; cell suffering ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The relationship between ribosome crystallization and cell degeneration has been studied in chick embryos at various temperatures, and new methods of inducing ribosome microcrystals are described. A model is discussed that reinterprets the role of low temperatures in these phenomena and provides a unitary explanation of the various cases in which the occurrence of ribosome crystallization in chick embryos has been reported.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 377-395 
    ISSN: 0091-7419
    Keywords: wet replicas ; microsurface spreading ; DNA ; subunits ; superbeads ; supercoils ; fibers ; chromosome bridges ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Superpacking of chromatin and the surface features of metaphase chromosomes have been studied by SiO replication of wet, unstained, and unfixed specimens in an exceedingly thin (≤ 1 nm) aqueous layer, keeping them wet. Hydrophilic Formvar substrates allow controlled thinning of the aqueous layer covering the wet specimens. Whole mounts of chromatin and chromosomes were prepared by applying a microsurface spreading method to swollen nuclei and mitotic cells at metaphase.The highest level of nucleosome folding of the inactive chromatin in chicken erythrocytes and rat liver nuclei is basically a second-order superhelical organization (width 150-200 nm, pitch distance 50-150 nm) of the elementary nucleosome filament. In unfavorable environments (as determined by ionic agents, fixative, and dehydrating agents) this superstructure collapses into chains of superbeads and beads. Formalin (10%) apparently attacks at discrete sites of chromatin, which are then separated into superbeads. The latter consist of 4-6 nucleosomes and seemingly correspond to successive turns of an original solenoidal coil (width 30-35 nm), which forms the superhelical organization. When this organization is unfolded, eg, in 1-2 mM EDTA, DNAse-sensitive filaments (diameter 1.7 nm) are seen to be wrapped around the nucleosomes.The wet chromosomes in each metaphase spread are held to each other by smooth microtubular fibers, 20-30 nm in diameter. Before they enter into a chromsome, these fibers branch into 9-13 protofilaments, each 5 nm wide. The chromosome surface contains a dense distribution of subunits about 10-25 nm in diameter. This size distribution corresponds to that of nucleosomes and their superbeads. Distinct from this beaded chromosome surface are several smooth, 23-30-nm-diameter fibers, which are longitudinal at the centromere and seem to continue into the chromatid structure. The surface replicas of dried chromosomes do not show these features, which are revealed only in wet chromosomes.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 14
    ISSN: 0091-7419
    Keywords: hepatoma ; cell culture ; tight junctions ; gap junctions ; freeze-fracture ; dexamethasone ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9-11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10-6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.
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  • 15
    ISSN: 0091-7419
    Keywords: cholera toxin ; GTP ; pigeon erythrocyte ; adenylate cyclase ; cytosolic factor ; phosphodiesterase protein activator ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The 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.
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  • 16
    ISSN: 0091-7419
    Keywords: lectin ; glycosaminoglycan ; extracellular material ; cell matrix ; cellular interactions ; myoblast development ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Embryonic chick muscle contains two developmentally regulated lectins, which may be involved in cell interactions. These endogenous lectins are assayed as agglutinins of appropriate test erythrocytes. One of these, called lectin-2, interacts with specific glycosaminoglycans, especially heparin and dermatan sulfate. Lectin-2 is present at constant levels in both chick fibroblast and chick muscle cells throughout 14 days of culture but is released into the medium of cultured embryonic muscle after 7-8 days of culture, soon after myoblast fusion. Lectin-2 interacts strongly with a component of substrate-attached material in embryonic muscle cultures which is extractable from the culture dishes with alkali after the cells have been removed with ethylediaminetetraacetic acid. The active component in the substrate-attached material appears to be a glycosaminoglycan that is a more potent inhibitor of lectin-2 agglutination activity than any of the known glycosaminoglycans that we have tested. The active material is degraded by chondroitinase ABC but not by chondrotinase AC, hyaluronidase, or proteolytic enzymes and thus appears to be similar to dermatan sulfate. The results of these studies raise the possibility that lectin-2 functions by interacting with glycosaminoglycans, either associated with the cell surface or with the extracellular matrix.
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  • 17
    ISSN: 0091-7419
    Keywords: cholera toxin ; chemical modification ; amino acid composition ; antigenic relationships ; radioimmunoassay ; adenylate cyclase ; ovine luteinizing hormone ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Chemical modification of intact cholera toxin or its B subunit by either partial nitration or reduction and alkylation did not result in significant loss of biological activity as determined by measurement of cyclic AMP in Chinese hamster overy cells. Complete nitration or succinylation in the presence of guanidine hydrochloride resulted in complete loss of biological activity and significantly affected the immunoreactivity of the toxin and B subunit. Compositional analyses of both the isolated α and γ chains of the toxin were typical of globular proteins and did not reveal significant hydrophobicity. Analysis of antigenic relationships by radioimmunoassay indicated a partial crossreactivity between the α chain and the B subunit of cholera toxin. Since previous structural studies of the β chain of cholera toxin indicated chemical similarity with the glycoprotein hormones [Kurosky et al. Science 195:299 (1977)], radioimmunoassay procedures were employed to investigate for possible crossreactivity. No evidence of crossreactivity between cholera toxin subunits and subunits of ovine luteinizing hormone was found.
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  • 18
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 151-163 
    ISSN: 0091-7419
    Keywords: NAD ; ADP-ribose ; poly ADP-ribose ; ADP-ribosyl protein ; cholera toxin ; adenylate cyclase ; pigeon erythrocyte ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Upon incubation of lysed pigeon erythrocytes with NAD, adenosine diphosphateribose (ADP-ribose) is incorporated into nuclear poly ADP-ribose and into an unidentified acid-insoluble product of the cytosol. The properties of these incorporations have been examined and a method developed for reducing their amount whilst retaining the sensitivity of the lysate to cholera toxin. This method has allowed the detection and description of a set of cholera toxin-specific ADP-ribose transfers to membrane-bound and soluble proteins under conditions that lead to adenylate cyclase activation.
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  • 19
    ISSN: 0091-7419
    Keywords: erythrocyte membranes ; LCAT deficiency ; electron spin resonance ; spin label ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different form that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physicochemical state of the membrane. An investigation of the spectral lineshapes below 10°C allowed a distinction to be made at the membarne level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of electron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.
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  • 20
    ISSN: 0091-7419
    Keywords: nascent chains ; co-translational modification ; glycosylation ; polypeptide folding ; covalent assembly ; heavy and light chains ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG2b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG1), where a heavy chain dimer is the major initial disulfide linked intermediate.
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  • 21
    ISSN: 0091-7419
    Keywords: cyclic AMP ; transport of nucleosides ; nucleobases ; hexoses ; Chinese hamster ovary cells ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a previous study we have demonstrated that neither extracellular nor intracellular cyclic adenosine monophosphate (AMP) levels directly affect the uptake of nucleosides, nucleobases, or hexoses by various types of cultured mammalian cells. Uptake of these nutrients into cells, however, involves two processes operating in tandem: facilitated transport across the membrane and intracellular phosphorylation; and uptake rates generally reflect the rates of substrate phosphorylation rather than of transport. In the present study we have examined the question of whether substrate transport per se is regulated by intracellular cyclic AMP. Initially various cell lines, grown both in suspension and monolayer culture, were screened for their cyclic AMP response to prostaglandin E1, isoproterenol, and inhibitors of cyclic AMP phosphodiesterase. Prostaglandin E1 treatment of Chinese hamster ovary cells was selected as the systems giving the largest and most consistent (50-fold to 100-fold) elevation of cyclic AMP. Rapid kinetic techniques were used to measure the transport of 3-O-methylglucose, thymidine, adenosine, hypoxanthine, and adenine in wild-type cells and in mutant sublines incapable of phosphorylating these substrates. In no case was an increse in intracellular cyclic AMP accompanied by a singinficant change in the rate of transport of these substrates, although prostaglandin E1 slightly inhibited the transport of various substrates.
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  • 22
    ISSN: 0091-7419
    Keywords: cell adhesion ; adhesion proteins ; fibronectin ; chondronectin ; collagen substrates ; gangliosides ; cell surface ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fibronectin mediates the adhesion of fibroblasts to collagen substrates, binding first to the collagen and then to the cells. We report here that the interaction of the cells with the fibronectin-collagen complex is blocked by specific gangliosides, GD1 a and GT1, and that the sugar moieties of these gangliosides contain the inhibitory activity. The gangliosides act by binding to fibronectin, suggesting that they may be the cell surface receptor for fibronectin. Evidence is presented that other adhesion proteins or mechanisms of attachment exist for chondrocytes, epidermal cells, and transformed tumorigenic cells, since adhesion of these cells is not stimulated by fibronectin. Chondrocytes adhere via a serum factor that is more temperature-sensitive and less basic than fibronectin. Unlike that of fibroblasts chondrocyte adhesion is stimulated by low levels of gangliosides. Epidermal cells adhere preferentially to type IV (basement membrane) collagen but at a much slower rate than fibroblasts or chondrocytes. This suggests that these epidermal cells synthesize their own specific adhesion factor. Metastatic cells cultured from the T241 fibrosarcoma adhere rapidly to type IV collagen in the absence of fibronectin and do not synthesize significant amounts of collagen or fibronectin. Their growth, in contrast to that of normal fibroblasts, is unaffected by a specific inhibitor of collagen synthesis. These data indicate the importance of specific collagens and adhesion proteins in the adhesion of certain cells and suggest that a reduction in the synthesis of collagen and of fibronectin is related to some of the abnormalities observed in transformed cells.
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  • 23
    ISSN: 0091-7419
    Keywords: fibronectin structure and properties ; cytoskeleton ; cell surface proteins ; fibronectin distribution ; fibronectin interactions ; transformation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000-dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotrpoic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.
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  • 24
    ISSN: 0091-7419
    Keywords: tumor metastases ; Fc receptor ; shedding ; tumor variants ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The expression of receptors for the Fc portion of IgG immunoglobin molecules was studied on tumor cell lines with high and low metastatic capacity. Two tumor cell lines from DBA/2 mice that had high metastatic activity, ESb and MDAY-D2, contained a high percentage of Fc receptor positive cells, as detected in a rosette assay with IgG antibody-coated erythrocytes (EA). In contrast, the low metastatic parental line Eb, from which ESb was derived, contained only a low percentage of EA-rosette-forming cells. ESb ascites tumor cells adapted to tissue culture in the presence of 2-mercaptoethanol (2ME) had a high expression of Fc receptors, whereas a cell line adapted to tissue culture in the absence of 2ME had a low expression of Fc receptors.“Soluble” Fc receptors were detectable by their ability to bind to EA and to cause blocking of rosette formation. They were found to be present in fluids from tumor-bearing animals, such as serum and cell-free ascites. Even animals with an ascites tumor of the low-metastatic line Eb contained “soluble” Fc receptors.The results are discussed with regard to their possible significance for tumor metastasis.
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  • 25
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 117-122 
    ISSN: 0091-7419
    Keywords: dynein ATPase ; latency ; high-affinity binding site ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The enhancing effect of low concentrations (eg, 8 μM) of bis(4-fluoro-3-nitrophenyl)sulfone (FNS) on 30S dynein ATPase activity is increased when 1 mM dithiothreitol (DTT) is present. The effect of FNS + DTT is optimal at pH 7.5. Activation of the latent ATPase activity of 30S dynein by FNS + DTT is partially prevented by 1-3 μM ATP. Adenylylimidodiphosphate (AMP-PNP) is less effective than ATP, while β,γ-methylene-adenosine triphosphate (AMP-PCP), though a much stronger inhibitor of ATPase activity than AMP-PNP, does not protect against enhancement. These results demonstrate the presence of a high-affinity ATP-binding site on 30S dynein.
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  • 26
    ISSN: 0091-7419
    Keywords: carcinoma and nonmalignant cells ; fibronectin ; human epithelial cells ; plasminogen activator ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human epithelial cell cultures were examined for expression of plasminogen activator and fibronectin matrix. All of the cells examined showed ultrastructural evidence suggesting their epithelial origin, including microvilli and specialized junctions. The nonmalignant cells were also negative for endothelial cell markers (ie, they lacked factor VIII antigen, a nonthrombogenic surface and Weibel-Palade bodies). The nonmalignant lines all produced large amounts of plasminogen activator, whereas the tumor-derived lines showed a gradation of activities, ranging from lines having as much activity as the nonmalignant lines to lines having little or no activity above background. For both normal and malignant cells, addition of dexamethesone only slightly decreased the levels of plasminogen activator. By immunofluorescence microscopy, normal bladder and fetal intestine epithelial cells showed fibronectin in a globular and fibrillar matrix. In contrast, normal mammary epithelial cells had a much diminished amount of fibronectin with a punctate distribution.
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  • 27
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 189-195 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The objective of this study was the preliminary characterization of the factors from mitotic HeLa cells that can induce meiotic maturation in Xenopus laevis oocytes. We found that this factor is a heat-labile, Ca2+-sensitive, nondialyzable protein with a sedimentation value of 4-5S. Furthermore, no new protein synthesis was found to be required for this mitotic factor to induce maturation in the amphibian oocytes. These data suggest that the factors involved in the breakdown of nuclear membrane and the condensation of chromosomes that are associated with three different phenomena, mitosis, meiosis, and premature chromosome condensation, are very similar in different animal species.
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  • 28
    ISSN: 0091-7419
    Keywords: growth control ; 3T3 cells ; Schwann cells ; neurites ; plasma membranes ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Control of cell growth by cell to cell contact is reviewed with particular emphasis on two systems - contact inhibition of growth observed with Swiss 3T3 cells and the mitogenic stimulation of Schwann cells by dorsal root ganglia neurites. In both cases the biological effect can be reproduced by the addition of surface membranes to the corresponding cells. In the case of contact inhibition of 3T3 cells, biological activity appears to correlate with membrane binding to the cells. An octylglucoside extract of 3T3 plasma membranes retains the biological activity (growth inhibition) of the original membranes.
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  • 29
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 197-205 
    ISSN: 0091-7419
    Keywords: pyrimidine biosynthesis ; V79 ; IARC19 ; IARC20 ; IARC28 ; biomarker ; pleiotropy ; confluence ; rat liver epithelial cells ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have developed procedures for sensitive measurement of specific radioactivities of pyrimidine nucleosides excreted from cells in culture. The changes in the observed values reflect dilution of the added isotope through de novo biosynthesis of nonradioactive pyrimidine nucleosides or by shifting and equilibration of other nucleotide pools into the free uridine pool. It is thus possible to monitor uridine biosynthesis occurring in intact cells without destroying or disrupting the cell population. On comparing a series of normal and transformed lines, we have observed several growth-dependent patterns of change in specific activity and levels of uridine excretion and the temporal appearance of these changes.Hamster embyro fibroblasts slows pyrimidine biosynthesis at mid-growth while the hamster cell line V79 continues to dilute the pyrimidine pool at about 7% of the rate observed during exponential growth at confluence. Both cells exhibit Urd excretion beginning at one-half maximal growth.Passageable normal rat liver cells (IARC-20) also show a cessation of pyrimidine biosynthesis with a prior increase in uridine excretion. Two chemically transformed lines IARC-28 and IARC-19 derived from IARC-20 show different patterns. IARC-19 begins uridine excretion in early log growth and the specific activity continues to decrease at about 2% of the rate observed during exponential growth at confluence. The IARC-28 cells also begin excretion in early log growth but pyrimidine biosynthesis stops at about midlog. This method may prove to be an additional aid in recognizing and differentiating transformed cells in culture that do not exhibit the transformed phenotype.
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  • 30
    ISSN: 0091-7419
    Keywords: fibronectin ; factor XIII ; transglutaminase ; collagen ; polyamine ; ∊(γ-glutamyl)-lysin ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Soluble fibronectin is found in body fluids and media of cultured adherent cells. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for factor XIIIa (plasma transglutaminase) and can be cross-linked to collagen and to the α chain of fibrin. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to investigate the possibility that factor XIIIa -mediated cross-linking is influenced by polyamines. Spermidine inhibited cross-linking between fibronectin and type I collagen, isolated α1 (I) collagen chains, or iodinated cyanogen bromide fragment 7 of α1 (I) chains (125I-α1 (I)-CB7). Half-maximal inhibition of crosslinking between 125I-α1 (I)-CB7 and fibronectin was observed when 0.1 mM spermine or spermidine was present. Spermidine, 0.7 mM, partially inhibited cross-linking between fibronectin and the α chain of fibrin but failed to inhibit cross-linking between the fibrin monomers of a fibrin clot. Spermidine also failed to inhibit cross-linking between fibronectin molecules when aggregation of fibronectin was induced with dithiothreitol. In contrast, 0.7 mM monodansylcadaverine inhibited fibronectin-collagen, fibronectin-fibrin, fibronectin-fibronectin, and fibrin-fibrin cross-linking. Spermidine or spermine, 0.7 mM, enhanced the cross-linking between molecules of partially amidinated fibronectin, suggesting that N1,8-(di-γ-glutamyl)-polyamine cross-linkages were formed. Spermidine and spermine failed to enhance cross-linking between monomers of amidinated fibrin. These results indicate that physiologic concentrations of polyamines specifically disturb transglutaminase-catalyzed cross-linking between fibronectin and collagen.
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  • 31
    ISSN: 0091-7419
    Keywords: tumorigenicity ; cyclic AMP-dependent protein kinase ; tyrosinase MSH-growth-resistant variant ; mouse melanoma ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A variant of B-16 F1 mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition by MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F1 cells. MSH induced a twofold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F1 cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F1 kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F1 and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F1 cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.
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  • 32
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    New York, N.Y. : Wiley-Blackwell
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 33
    ISSN: 0091-7419
    Keywords: cell surface receptors ; proteolysis of receptors ; positive or negative regulation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Addition of highly purified thrombin t o cultures of several kinds of nondividing fibroblasts brings about cell division. This stimulation occurs in serum-free medium, permitting studies on its mechanism under chemically defined conditions. Previous studies have shown that action of thrombin a t the cell surface is sufficient to cause cell division and that the proteolytic activity of thrombin is required for its mitogenic effect. These results prompted experiments which showed that there is a cell surface receptor for thrombin and that thrombin must hind to its receptor and cleave it to stimulate cell division. Some of the thrombin that hinds to its receptors becomes attached to them by a linkage that appears to be covalent. However, it is presently unknown whether this direct thrombin receptor complex plays a role in the stimulation.These results raise a number of question that should be explored in future studies. They also provide a foundation on which to build hypotheses about tentative molecular mechanisms that might be involved in the stimulation. Knowledge that thrombin must cleave its receptor to bring about cell division suggests two alternative mechanisms for stimulation by proteolysis. In one the receptor is a negative effector which prevents cell division when it is intact, but not after it has been cleaved. Alternatively, a fragment of the receptor could be a positive effector. In this mechanism, proteolysis by thrombin would produce a specific receptor fragment which brings about cell proliferation. If every protease which cleaves the receptor also stimulates cell division, the receptor is probably a negative effector. In contrast, if certain proteases cleave the receptor but do not stimulate the cells, a fragment of the receptor is likely a positive effector. With negative regulation by the receptor, the controlling events would occur before proteolysis of it, and it might be possible to find putative regulatory molecules by identification of nearest neighbors of the receptor. This should be possible by using bifunctional crosslinking reagents. If a fragment of the thrombin receptor turns out to be a positive effector, it should be possible to identify and study fragments by analyzing the metabolic fate of the receptor. Techniques are now available for this kind of analysis and it should also be possible to determine whether receptor fragments remain in the membrane or whether they are translocated to specific sites within the cell. A critical question to be asked is which of these events and interactions involving the thrombin receptor are necessary for stimulation of cell division. It now appears that the best way to answer this question is to examine these events in a large number of cloned cell populations that are responsive or unresponsive to the mitogenic action of thrombin. If a thrombin-mediated event occurs in all responsive clones but is altered or absent in sonie unresponsive clones, it is probably necessary for stimulation of cell division.
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  • 34
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 283-293 
    ISSN: 0091-7419
    Keywords: cytochalasins ; muscle and platelet actin ; microfilaments ; cell motility ; viscosity changes ; electron microscopy ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The cytochalasins (CE, CD, CB and H2CB) inhibit numerous cellular processes which require the interaction of actin with other structural and contractile proteins. In this report we describe the effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin. The cytochalasins decreased the viscosity of F-actin solutions. The effect of H2CB, CB and CD on F-actin viscosity was maximal at concentrations of 20-50μM and did not increase with time. In contrast, CE caused a progressive decrease in the viscosity of F-actin solutions which was dependent upon the concentration of CE and the duration of incubation of the CE-actin mixture. After two hours of incubation of drug-actin mixtures, the relative effectiveness of the cytochalasins in reducing the viscosity of F-actin was CE 〉 CD 〉 CB = H2CB. The effects of CD and CE were paralleled by morphologic changes in negatively stained actin filaments. The effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin were the same whether the drugs were added before or after the polymerization of the protein.These studies show that the interaction of the cytochalasins with actin is highly specific. Because the relative potencies of these drugs for affecting motile processes and the relative affinities of the drugs for binding sites within a variety of cells are CE 〉 CD 〉 CB = H2CB, the effects of cytochalasins on actin described here may contribute to some of the biological effects of the drugs on motile processes.
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  • 35
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    Journal of Supramolecular Structure 11 (1979), S. 311-317 
    ISSN: 0091-7419
    Keywords: dynein ; flagella ; ATPase ; sperm motility ; sea urchin ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000-350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4°C with a solution containing 0.6 M NaCl, pH 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21 S and a minor peak at 12-14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000-122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000-24,000) cosediment with the 21 S peak. The heavy chain composition of the 12-14S peak is more complex, all eight heavy chains occurring in approximately the same ratios as occur in intact axonemes.
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  • 36
    ISSN: 0091-7419
    Keywords: AcChR-enriched membranes ; pyrenesulfonyl azide ; fluorescent probes ; photolabeling ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene-1-sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core.Covalent attachment of the photogenerated fluorescence probe does not perturb the α-neurotoxins' binding properties of membrane-bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non-covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra or fluorescence lifetimes, are highly sensitive to the solvent milieu. The covalently bound probe shows similar fluorescence properties in situ. PySA photoproducts have great potential to spectroscopically monitor neurotransmitter induced events on selected AcChR subunits exposed to the hydrophobic environment of membranes.
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  • 37
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    Journal of Supramolecular Structure 11 (1979), S. 349-359 
    ISSN: 0091-7419
    Keywords: colostrum ; milk ; serum ; growth factors ; mitogens ; DNA synthesis ; proliferation ; 3T3 cells ; serum-free growth ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bovine milk contains growth promoting factors that stimulate DNA synthesis and cell division in confluent monolayers of quiescent Balb/c 3T3 cells. The growth factor activity was highest in colostrum obtained within 24 hours after birth of a calf. Samples of milk obtained 32 hours and 60 hours after birth were 20% and 1% as active respectively as was a sample obtained 8 hours after birth in stimulating DNA synthesis. No activity was detectable 3 days after birth or thereafter. A similar temporal dependence was found in sheep's milk. Bovine colostrum obtained on the day of a calf's birth can be substituted for serum and will support the growth of sparse Balb/c 3T3 cells to confluence. In Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% (vol/vol) bovine colostrum, the number of Balb/c 3T3 cells in a dish increased 35-fold, from 2.0 × 104 cells to 7 × 105 cells. The generation time was approximately 38 hours. Proliferation of cells was characterized by formation of clusters of confluent Balb/c 3T3 cells which were smaller in size and more tightly packed than were Balb/c 3T3 cells grown to confluence in serum. No proliferation was detected in DMEM supplemented with milk obtained 10 days after birth of a calf or in DMEM supplemented with bovine serum albumen.
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  • 38
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    New York, N.Y. : Wiley-Blackwell
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 39
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 391-399 
    ISSN: 0091-7419
    Keywords: cold-insoluble globulin ; carbohydrate structure ; human plasma ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cold-insoluble globulin (CIg) is a member of a group of circulating and cell-associated, high-molecular-weight glycoproteins termed fibronectins. CIg was isolated from human plasma by affinity chromatography on gelatin-Sepharose. SDS-polyacrylamide gel electrophoresis of the purified glycoprotein gave a double band that migrated near myosin. The CIg glycopeptides were released by pronase digestion and isolated by chromatography on Sephadex G-50. Affinity chromatography of the major G-50 peak on Con A-Sepharose resulted in two fractions: one-third of the glycopeptides were unbound and two-thirds were weakly bound (WB). Sugar composition analysis of the unbound glycopeptides by GLC of the trimethylsilyl methyl glycosides gave the following molar ratios: sialic acid, 2.5; galactose, 3.0; N-acetylglucosamine, 4.9; and mannose, 3.0. Sugar composition analysis of the WB glycopeptides gave the following molar ratios: sialic acid, 1.7; galactose, 2.0; N-acetylglucosamine, 4.1; and mannose, 3.0. The WB CIg glycopeptides cochromatographed on Sephadex G-50 with WB transferrin glycopeptides giving an estimated molecular weight of 2,800. After degradation with neuraminidase alone or sequentially with β-galactosidase the CIg and transferrin glycopeptides again cochromatographed. Methylation linkage analysis of the intact and the partially degraded glycopeptides indicated that the carbohydrate structure of the major human CIg glycopeptide resembles that of the major glycopeptide from transferrin.
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  • 40
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    Journal of Supramolecular Structure 11 (1979), S. 445-449 
    ISSN: 0091-7419
    Keywords: subcellular fractionation ; brown adipose tissue ; plasma membranes ; microsomes ; Metrizamide ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The present study proposes a technique, using Metrizamide, which permits the preparation of brown adipose tissue plasma membranes from the crude mitochondria as well as from the crude microsome fraction. These plasma membranes have high relative specific activities of their marker enzyme, 5′-nucleotidase (15 ± 3 and 14 ± 2 respectively) and, particularly those originating in the crude microsomes, are relatively free of mitochondria contamination. This study also shows the influence of the mode of cell disruption on microsome integrity. When cell disruption was achieved by grinding in liquid nitrogen the purified microsome NADPH cytochrome c reductase specific activity was found to be 3.5 times greater than that of microsomes obtained after homogenization of the tissue.
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  • 41
    ISSN: 0091-7419
    Keywords: alkaline phosphatase ; basal lateral membranes ; brush border membranes ; intestinal epithelium ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The alkaline phosphatases present on isolated brush border and basal lateral membranes of rat duodenal epitheilum were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6-9.8, similar substrate km's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin or papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000-150,000, and both enzymes show an Rf value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The Vmax of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 μmoles/mg-h as opposed to 14 μmoles/mg-h. The differences in Vmax are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.
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  • 42
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    Journal of Supramolecular Structure 12 (1979), S. 115-125 
    ISSN: 0091-7419
    Keywords: glycocalyx ; cell surface ; tumor cells ; Ehrlich ascites tumor ; surface labeling ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4′-diisothiocyano-1,2′-dihenylethane-2,2′-disulfonic acid (3H2DIDS) at 4°C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of 3H2DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the 3H2DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23°C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (τ½ = 360 min) component representing 33% of the radioactivity, and a fast (τ½ = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 μ/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinectics of release to that of a single component (τ½ = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously los in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither 3H2DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.
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  • 43
    ISSN: 0091-7419
    Keywords: fibronectin ; CSP-60 ; extracellular matrix ; thrombogenic properties ; low-density lipoprotein ; receptor redistribution ; asymmetry of cell surfaces ; cell morphology ; spatial configuration ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vascular endothelial cells cultured in the presence of fibroblast growth factor (FGF) devide actively when seeded at low or clonal cell densities and upon reachin confluence adopt a morphologic appearance and differentiated properties similar to those of the vascular endothelium in vovi. In this review, we present some of our recent observations regarding the characteristics (both structural and functional) of these endothelial cells and the role of FGF in controlling their proliferation and normal differentation. At confluence the endothelial cells from a monolayer of closely apposed and nondividing cell that have a nonthrombogenic apical surface and can no longer internalize bound ligands such as low-density lipoprotein (LDL). The adoption of these properties is correlated and possibly causally related to changes in the cell surface such as the appearance of a 60,000 molecular weight protein (CSP-60); the disappearance of fibronectin from the apical cell surface and its concomitant accumulation in the basal lamina; and a restriction of the lateral mobility of various cell surface receptor sites. In contrast, endothelial cells that are maintained in the absence of FGF undergo within three passages alterations that are incompatible with their in vivo morphologic apperarance and physiologic beharior. They grow at confluence on top of each other and hence can no longer adopt both the structural (CSP-60, cell surface polarity) and functional (barrier function, nonthrombogenicity) attributes of differentiated endothelial cell. Since these characteristics can be reacquired in response to readdition of FGF, in addition to being a mitogen FGF may also be involved in controlling the differentitation and phenotypic expression of the vascular endothelium.
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  • 44
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 139-150 
    ISSN: 0091-7419
    Keywords: fibronectin ; tumor ; malignancy ; cell shape ; hormone ; embryogenesis ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Frozen sections of tumors induced by injecting virally transformed cells into animals were stained for fibronectin by immunofluorescence. Many tumor cell lines do not express fibronectin in tumors in situ even though some of them express fibronection in culture. Cell shape and hormones appear to influence the expression of fibronectin in culture; however, it is nuclear how fibronection expression is regulated in vivo.
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  • 45
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    New York, N.Y. : Wiley-Blackwell
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
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  • 46
    ISSN: 0091-7419
    Keywords: denervated sarcolemma ; nonsynaptic acetylcholine receptors ; 125I-α-bungarotoxin ; ferritin-α-bungarotoxin ; electron microscopy ; freeze-fracture ; freeze-etching ; autoradiography ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nonsynaptic sarcolemma of denervated skeletal muscle of rat shows an abundance of ∼15 nm intramembranous particles on the P face. These particles are either singly distributed or are in clusters, and they are essentially lacking from the comparable freeze-fractures of the innervated sarcolemma. Autoradiographic studies using 125I-α-bungarotoxin (BGT) on 1 μ-thick sections, and freeze-etch studies using ferritin-α-BGT conjugates on membrane fractions, show that the distribution of the label corresponds to the distribution of the 15-nm particles in the nonsynaptic sarcolemma. On the basis of these results and existing physiologic and biochemical data, it is suggested that the 15-nm intramembranous particles are components of the α-BGT binding sites, ie, acetylcholine (Ach) receptors, in the nonsynaptic sarcolemma of denervated muscle and that the two types of distributions represent two spatial manifestations of Ach receptor molecules. The significance of these findings in relation to synapse formation in denervated muscle is discussed.
    Additional Material: 5 Ill.
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  • 47
    ISSN: 0091-7419
    Keywords: Alkaline phosphatase ; chromosome-mediated gene transfer ; human breast tumor cells ; hydrocortisone ; lipochromes ; membrane bound enzymes ; nucleoside uptake ; thymidine kinase ; thymidine transport ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 X 10-5 and 1 X 10-5, respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and survives on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 μM hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.
    Additional Material: 4 Ill.
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  • 48
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
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  • 49
    ISSN: 0091-7419
    Keywords: hybrid resistance ; NK cells ; cytotoxic lymphocytes ; tumor-associated antigens ; primary and metastatic tumor cells ; immunoselection ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The existence of antigenic differences between cell populations in the local growth of the 3LL tumor (L-3LL) and its lung metastases (M-3LL) was studied. Normal C57BL/6 spleen cells sensitized in vitro for 5 days against L-3LL monolayers lysed preferentially L-3LL targets but not M-3LL tumor cell targets. Conversely, anti-M-3LL-sensitized lymphocytes killed M-3LL targets more efficiently than they killed L-3LL targets. Furthermore, spleen cells from mice bearing subcutaneous L-3LL tumors were significantly more cytotoxic to L-3LL targets than to M-3LL targets and vice versa. M-3LL cells were found also to be more resistant in vitro and in vivo to natural killer cells than were L-3LL tumor cells. M-3LL cells were more resistant than L-3LL cells to hybrid resistant mechanisms when they were inoculated into F1 (C3Heb X C57BL/6) or F1 (BALB/c X C57BL/6) mice. Anti-M-3LL lymphocytes generated both in vitro and in vivo, but not anti-L-3LL lymphocytes, admixed with L-3LL or M-3LL tumor cells and inoculated into footpads of syngeneic recipients suppressed the development of lung metastases. These results suggest that metastatic cells are indeed phenotypic variants of the local growing tumor cell populations. Presumably, these variants are selected for their capacity to home to and grow in the lungs, and for their resistance to specific immune effects initially evoked against the local tumor and to nonspecific natural killer cells. These data may prove to be of importance with respect to any rational approach to the problem of immunotherapy.
    Additional Material: 4 Ill.
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  • 50
    ISSN: 0091-7419
    Keywords: direct labeling of EGF receptors ; transient down-regulation of EGF receptors ; platelet derived growth factor ; receptor proteolysis ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A proposal that EGF action is mediated through enhanced internalization of EGF receptors is modified to account for more recent evidence. EGF receptors turn over at a rapid rate, and the maintenance of a steady state of EGF receptors on the cell surface is provided through a rapid synthesis of EGF receptors, balancing their removal. This rapid turnover of unoccupied receptors may arise through their removal. This rapid turnover of unoccupied receptors may arise through their internalization and proteolysis in the lysosomes, in much the same way as receptors are internalized and degraded when exposed to EGF, which enhances internalization. This provides a dilemma for the endocytic activation concept, since slight enhancement of receptor internalization gives rise to a strong hormone response. This problem may be solved by the observation that EGF induces a change in its receptor, exposing an otherwise unavailable site for proteolytic cleavage. This hormone-dependent modification of receptors may be the critical step in the induction of responses to EGF and other hormones that are internalized with their receptors. Both platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are shown to down-regulate EGF receptors, though transiently, placing still more stringent requirements on the specificity by which hormones might act through endocytic activation of their receptors.
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  • 51
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 52
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 121-160 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 53
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 161-231 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 54
    ISSN: 0091-7419
    Keywords: lectins ; binding sites ; neuroblastoma cells ; receptor redistribution ; cell surface labeling ; cytochalasin B ; concanavalin A ; wheat germ agglutinin ; fluorescent microscopy ; scanning electron microscopy ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Concanavalin 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.
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  • 55