Functional incorporation and preferred orientation of phytohemagglutinin receptor glycoproteins in phospholipid vesicles

1987 ◽  
Vol 65 (2) ◽  
pp. 120-129 ◽  
Author(s):  
Gilles Dupuis ◽  
Marc Letellier
Keyword(s):  
Cultured Cells ◽  
Lymphocyte Activation ◽  
Lipid Vesicles ◽  
Simultaneous Action ◽  
Phospholipid Vesicles ◽  
Lectin Receptor ◽  
Low Concentrations ◽  
Binding Data ◽  
Combined Action ◽  
Tritium Labeling

Porcine lymphocyte Phaseolus vulgaris phytohemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography have been reassembled into vesicles made of phosphatidylcholine and phosphatidylserine by detergent (dodecyltrimethylammonium bromide) dialysis. The receptor glycoproteins were incorporated into the lipid vesicles in a nonselective manner with a yield of 65–70%. Vesicles containing the glycoproteins were sealed as evidenced by their impermeability to calcium ions, using quin 2 trapped inside the vesicles. The vesicles were agglutinated by PHA, suggesting that the saccharidic moiety of the reconstituted glycoproteins was, at least in part, oriented towads the extravesicular medium. This observation was further supported by the fact that the vesicles bound 125I-labeled PHA in a specific and saturable manner. At maximum amount of lectin bound, a ratio of 1.01 ± 0.05 μg of PHA per microgram glycoprotein incorporated was measured. When the binding data were analyzed by Scatchard plot, a downward concave profile was observed, suggestive of a positive cooperativity at low concentrations of lectin. The orientation of the reconstituted lectin receptor glycoproteins was determined by proteolytic treatments of labeled glycoproteins. The combined action of trypsin and chymotrypsin released, in the 120 000 × g supernatant, approximately 80% of label when 125I-tagged PHA receptor glycoproteins were incorporated into the vesicles. When the oligosaccharidic moieties of the receptor glycoproteins were specifically labeled, the simultaneous action of the two enzymes released approximately 70% of tritium labeling present in the reconstituted system. Taken together, these results suggest that the reconstituted PHA receptors are preferentially oriented into the phospholipid vesicles. The reconstituted PHA receptor glycoproteins competed effectively with cellular receptors in the assay of PHA-induced porcine lymphocyte activation. A 50% inhibition of [3H]thymidine incorporation was observed when 1 μg of glycoproteins in vesicles was added to the cultured cells, whereas vesicles alone had no effect at this (equivalent) concentration.

PRODUCTION AND CHARACTERISATION OF A MONOCLONAL ANTIBODY AGAINST HUMAN ANTI-THROMBIN III

1987 ◽  
Author(s):  
Deborah A Rathjen ◽  
Carolyn L Geczy
Keyword(s):  
Cultured Cells ◽  
Factor Xa ◽  
Lymphocyte Activation ◽  
Blue Staining ◽  
Aortic Endothelial Cells ◽  
Tissue Sections ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
At Iii

To study the role of anticoagulants, particularly antithrombin III (AT III) and heparin, on the activation of coagulation by monocytes/macrophages which have been stimulated with a soluble lymphocyte activation product, macrophage procoagulant inducing factor, we have prepared monoclonal antibodies (MAbs) to human AT III.In fusion experiments, in contrast to wells containing peritoneal feeder cells, positive hybrids were only found in wells containing medium conditioned by the macrophage cell line J774 (Rathjen and Geczy, 1986). Of 5 hybrids which initially produced antibody, only one hybrid, showed stable Ab production. The MAb, designated 22/23, was not cross-reactive with either 1 antitrypsin or ovalbumin and did not inhibit the biological activity of AT III in chromogenic assays which measured inhibition of thrombin and Factor Xa by AT III. An immunoadsorbent prepared using MAb 22/23 depleted AT III activity from a purified AT III preparation. Reduction and alkylat ion of the disulphide bonds of the protein portion of AT III completely abbrogated MAb binding indicating that the native configuration of AT III was important. Isoelectric focussing of AT III, followed by transfer of the focussed protein to nitrocellulose by diffusion and probing with MAb 22/23, revealed at least 8 bands in the region of pH 5.2 to 5.85. Coomassie blue staining of a gel run in parallel showed 9 bands in this region. The MAb provides a useful tool for the detection of AT III on both cultured cells (bovine aortic endothelial cells are positive by immunofluorescence) and tissue sections.Rathjen, D.A. and Geczy, C.L. Hybridomo. 5s 255-261 (1986)

scholarly journals The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid

1998 ◽  
Vol 180 (24) ◽  
pp. 6565-6570 ◽  
Author(s):  
Gert N. Moll ◽  
Wil N. Konings ◽  
Arnold J. M. Driessen
Keyword(s):  
Antimicrobial Peptide ◽  
Rate Constant ◽  
Lipid Vesicles ◽  
Phospholipid Vesicles ◽  
Nisin Concentration ◽  
Outer Leaflet ◽  
Target Membrane

ABSTRACT Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant k of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30°C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.

scholarly journals Specific identification of fibrin polymers, fibrinogen degradation products, and crosslinked fibrin degradation products in plasma and serum with a new sensitive technique

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 589-597
Author(s):  
DG Connaghan ◽  
CW Francis ◽  
DA Lane ◽  
VJ Marder
Keyword(s):  
Degradation Products ◽  
Sodium Dodecyl ◽  
Fibrinolytic Therapy ◽  
Electrophoretic Patterns ◽  
Fibrin Degradation Products ◽  
Low Concentrations ◽  
Combined Action ◽  
Derivatives Of ◽  
Normal Controls ◽  
Immunological Identification

A new method is described for identifying low concentrations of circulating derivatives of fibrinogen and fibrin, even when present in heterogeneous mixtures. This technique is applicable to plasma and serum and uses electrophoresis in 2% agarose in the presence of sodium dodecyl sulfate (SDS) followed by immunological identification of separated derivatives, using radiolabeled antifibrinogen antiserum and autoradiography. Unique electrophoretic patterns distinguish plasmic derivatives of crosslinked fibrin from those of fibrinogen and also identify crosslinked fibrin polymers produced by the combined action of thrombin and factor XIII on fibrinogen. The assay is sensitive to a concentration of 0.1 micrograms/mL of fibrinogen in serum or plasma. Fibrin polymers, plasmic degradation products of fibrinogen, and plasmic degradation products of crosslinked fibrin were detected in the plasma or serum of a patient with disseminated intravascular coagulation. Plasmic derivatives of both fibrinogen and crosslinked fibrin appeared in serum in the course of fibrinolytic therapy for pulmonary embolism, whereas during acute myocardial infarction a marked increase in the proportion of fibrin polymers in plasma was found in comparison with normal controls. Thus, the procedure can distinguish between the simultaneous processes of fibrin polymer formation, fibrinogenolysis, and fibrinolysis, and is sufficiently sensitive to detect relevant quantities of derivatives in pathologic conditions.

Modelling the adsorption of phospholipid vesicles to a silicon dioxide surface using Langmuir kinetics

2022 ◽  
Author(s):  
Iad Alhallak ◽  
Peter J. N. Kett
Keyword(s):  
Silicon Dioxide ◽  
Equilibrium Constant ◽  
Rate Constants ◽  
Lipid Vesicles ◽  
Phospholipid Vesicles ◽  
Adsorption And Desorption ◽  
Langmuir Kinetics

The rate constants and equilibrium constant for the adsorption and desorption of lipid vesicles from a SiO2 surface have been determined.

Characterization and Properties of Different Glucosyltransferases Isolated from Suspension-Cultured Cells of Daucus carota

1986 ◽  
Vol 41 (4) ◽  
pp. 409-420 ◽  
Author(s):  
Edgar Ingold ◽  
Hanns Ulrich Seitz
Keyword(s):  
Daucus Carota ◽  
Cultured Cells ◽  
Test System ◽  
Enzymatic Activities ◽  
Daucus Carota L ◽  
Low Concentrations ◽  
Nucleotide Sugars ◽  
Glucan Chain ◽  
Glucan Synthesis

Particulate enzymes (14,000 g pellet) from suspension-cultured carrot cells (Daucus carota L.) incorporated glucose from UDP-glucose and GDP-glucose into ethanol-insoluble products which were characterized as glucans or glucoprotein. Based on the test system to assay glucansynthe- tases I and II four different enzymatic activities could be distinguished on the basis of their substrate and divalent cation requirements, the influence of active substances such as nucleotides, nucleotide sugars, cellobiose, and in vivo inhibitors of cell wall glucan synthesis, their distribution in linear sucrose gradient and the nature of their products. The enzymatic activities which incor­porated glucose from UDP-glucose or GDP-glucose at low substrate concentrations (10 -6 ᴍ) were both localized in membranes of a density of 1.129 g em-3 (Golgi membranes) and synthesized a β-1,4-glucan chain. Both showed similar properties in most of the characterization experiments. The glucosyltransferase that catalysed the formation of a β-1,3-glucan from UDP-glucose (0.48 mᴍ) was found in membranes which accumulated at a density of 1.170 g · cm-3 (plasma membrane) and differed in its properties from the Golgi-localized glucosyltransferase activities in many aspects. A soluble glucosyltransferase (175,000 × g supernatant) which was also active at low concentrations of UDP-glucose (10-6 ᴍ) but showed enhanced activity under conditions where the other glucosyltransferases were inactive incorporated glucose into a proteinase-sensi­tive product. In linear sucrose gradients this enzyme migrated to different gradient densities depending on conditions.

scholarly journals Effect of sodium butyrate on lymphocyte activation

1976 ◽  
Vol 144 (6) ◽  
pp. 1674-1678 ◽  
Author(s):  
D Kyner ◽  
P Zabos ◽  
J Christman ◽  
G Acs
Keyword(s):  
Tissue Culture ◽  
Dna Synthesis ◽  
Sodium Butyrate ◽  
Cell Morphology ◽  
Mammalian Cells ◽  
Lymphocyte Activation ◽  
Extended Period ◽  
Present Evidence ◽  
Growth Of A Variety ◽  
Low Concentrations

Butyrate, in relatively low concentrations, has been shown to induce synthesis of enzymes, cause changes in cell morphology, and inhibit growth of a variety of mammalian cells in tissue culture (reviewed in [1]). In this communication, we report our observations on the effect of butyrate on lymphocyte activation. Butyrate completely and reversibly inhibits mitogen-induced blast formation. We present evidence that it does not interfere with the binding of mitogens, that it does not inhibit a number of the early reactions involved in activation, and that it does not affect ongoing DNA synthesis for an extended period of time. However, butyrate rapidly inhibits any increase in the rate of DNA synthesis.

Reconstitution of the complement channel into lipid vesicles and planar bilayers starting from the fluid phase complex

1985 ◽  
Vol 5 (2) ◽  
pp. 129-136 ◽  
Author(s):  
Gianfranco Menestrina ◽  
Flavia Pasquali
Keyword(s):  
Lipid Bilayers ◽  
Membrane Attack Complex ◽  
Fluid Phase ◽  
Lipid Vesicles ◽  
Ionic Channels ◽  
Single Channels ◽  
Planar Bilayers ◽  
Low Concentrations ◽  
Host Membrane ◽  
Phase Complex

Proteolysis of the fluid phase complement complex SC5b-9 transforms it into an arnphiphilic molecule which resembles the membrane attack complex of complement and reconstitutes into lipid vesicles. Complement-containing vesicles prepared in this way can be made to fuse with planar lipid bilayers transferring their protein content to the host membrane. Massive conductance increases can thus be observed, which are due to the insertion of a large number of ionic channels into the membrane. Using low concentrations of vesicles, single channels can be studied.

scholarly journals A combined action of pulmonary surfactant proteins SP-B and SP-C modulates permeability and dynamics of phospholipid membranes

2011 ◽  
Vol 438 (3) ◽  
pp. 555-564 ◽  
Author(s):  
Elisa Parra ◽  
Lara H. Moleiro ◽  
Ivan López-Montero ◽  
Antonio Cruz ◽  
Francisco Monroy ◽  
...  
Keyword(s):  
Pulmonary Surfactant ◽  
External Medium ◽  
Nile Red ◽  
Lipid Vesicles ◽  
Membrane Lipid ◽  
Structure And Dynamics ◽  
Lipid Dynamics ◽  
Combined Action ◽  
Surface Active ◽  
Pulmonary Surfactant Proteins

Proteins SP-B and SP-C are essential to promote formation of surface-active films at the respiratory interface, but their mechanism of action is still under investigation. In the present study we have analysed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of the fluorescent probes Nile Red (permeable) and FM 1-43 (impermeable) into membranes. We have also analysed the effect of single or combined proteins on membrane permeation using the soluble fluorescent dye calcein. The fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast with Nile Red which was very similar in all of the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C caused giant oligolamellar vesicles to incorporate FM 1-43 from the external medium into apparently most of the membranes instantaneously. In contrast, oligolamellar pure lipid vesicles appeared to be mainly labelled in the outermost membrane layer. Pure lipidic vesicles were impermeable to calcein, whereas it permeated through membranes containing SP-B and/or SP-C. Vesicles containing only SP-B were stable, but prone to vesicle–vesicle interactions, whereas those containing only SP-C were extremely dynamic, undergoing frequent fluctuations and ruptures. Differential structural effects of proteins on vesicles were confirmed by electron microscopy. These results suggest that SP-B and SP-C have different contributions to inter- and intra-membrane lipid dynamics, and that their combined action could provide unique effects to modulate structure and dynamics of pulmonary surfactant membranes and films.

scholarly journals Selective Irreversible Inactivation of Replicating Mengovirus by Nucleoside Analogues: a New Form of Viral Interference

1999 ◽  
Vol 73 (8) ◽  
pp. 6444-6452
Author(s):  
Branko Brdar ◽  
E. Reich
Keyword(s):  
Rna Synthesis ◽  
Cultured Cells ◽  
Virus Production ◽  
Adenosine Kinase ◽  
Nucleoside Transport ◽  
Nucleoside Analogues ◽  
Virus Growth ◽  
Development Cycle ◽  
Low Concentrations ◽  
Infected Cells

ABSTRACT We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 μg/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 μg/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 μg/ml) and Tu (1 to 20 μg/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus “cured” of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection.

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