scholarly journals Intralumenal pool and transport of CMP-N-acetylneuraminic acid, GDP-fucose and UDP-galactose. Study with plasma-membrane-permeabilized mouse thymocytes

1984 ◽  
Vol 224 (1) ◽  
pp. 277-284 ◽  
Author(s):  
R Cacan ◽  
R Cecchelli ◽  
B Hoflack ◽  
A Verbert
Keyword(s):  
Plasma Membrane ◽  
Isotopic Dilution ◽  
Saturation Curve ◽  
Sugar Nucleotides ◽  
Acetylneuraminic Acid ◽  
Substrate Analogues ◽  
Carrier Mediated Transport ◽  
Carrier Molecule ◽  
Substrate Saturation

Treatment with NH4Cl of mouse thymocytes renders their plasma membrane permeable to sugar nucleotides both inwards and outwards. Using this model, we studied the entry and utilization of CMP-NeuAc, GDP-Fuc and UDP-Gal into intracellular vesicles in situ. It is shown that CMP-NeuAc and GDP-Fuc enter the vesicles in a manner indicating a carrier-mediated transport (substrate saturation curve, inhibition by substrate analogues, temperature dependence) and are entrapped in their uncleaved form. This leads to the formation of an intralumenal pool of these precursors which can be further utilized by the sialyltransferases and fucosyltransferases. The occurrence of an endogenous pool of CMP-NeuAc and GDP-Fuc is demonstrated by the fact that, when the vesicles are disrupted by detergent, the release of the endogenous sugar nucleotides causes an isotopic dilution of the labelled precursors added to measure the glycosyltransferase activities. In contrast, no accumulation of UDP-Gal has been detected, suggesting that transport and transfer reaction are simultaneous events. However, experiments with UDP 2′,3′-dialdehyde indicate that UDP-Gal is not transported through the membrane by galactosyltransferase action but by a distinct carrier molecule.

scholarly journals Glycosylation of proteins from sugar nucleotides by whole cells. Effect of ammonium chloride treatment on mouse thymocytes

1983 ◽  
Vol 216 (3) ◽  
pp. 681-686 ◽  
Author(s):  
R Cecchelli ◽  
R Cacan ◽  
B Hoflack ◽  
A Verbert
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Trypan Blue ◽  
Sugar Nucleotides ◽  
Whole Cells ◽  
Acetylneuraminic Acid ◽  
Intracellular Vesicles ◽  
Chloride Treatment ◽  
Stimulation Of

When thymocytes are treated with iso-osmotic NH4Cl, the sugar incorporation into endogenous acceptors from labelled sugar nucleotides is largely increased compared with that in control thymocytes. This effect was obtained with labelled GDP-mannose, UDP-galactose and CMP-N-acetylneuraminic acid. The stimulation observed with NH4Cl-treated thymocytes does not involve the glycosylation of exogenous acceptors, and it was proved that the NH4Cl treatment (1) does not stimulate glycosyltransferase activities themselves, (2) does not lead to the release of soluble glycosyltransferases as the result of an extensive lysis of the thymocytes and (3) does not cause the emergence of glycosyltransferases at the cell surface. In fact, electron-microscopy observations showed that, although marked changes had occurred in the cytoplasm, the plasma membrane is sufficiently maintained to allow the cell to keep roughly its original shape and to retain the intracellular vesicles. We thus demonstrate that this stimulation is due to an enhancement of the entry of sugar nucleotides into the cell. As demonstrated by the inclusion of Trypan Blue within the cells, and the non-stimulation of glycosylation of exogenous large-molecular-mass acceptors, the effect of NH4Cl seems to be limited to the penetration of small-molecular-sized compounds through the plasma membrane. Thus NH4Cl treatment allows the labelled sugar nucleotides to penetrate the cell and to behave as the cellular pool to be utilized for glycosylation by intracellular vesicles.

scholarly journals A cell-free assay for the insertion of a viral glycoprotein into the plasma membrane.

1986 ◽  
Vol 103 (5) ◽  
pp. 1829-1835 ◽  
Author(s):  
P G Woodman ◽  
J M Edwardson
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Trichloroacetic Acid ◽  
Plasma Membranes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Viral Glycoprotein ◽  
Acetylneuraminic Acid ◽  
Donor And Acceptor ◽  
A Cell

A cell-free assay has been developed for the delivery of influenza virus neuraminidase to the plasma membrane. Two types of postnuclear supernatant, which acted as donor and acceptor of the enzyme, were prepared from baby hamster kidney cells. Donor preparations were obtained from cells infected with influenza virus and containing neuraminidase en route to the plasma membrane. Acceptor preparations were obtained from cells containing, bound to their plasma membranes, Semliki Forest virus with envelope glycoproteins bearing [3H]N-acetylneuraminic acid. Fusion between vesicles from these two preparations permits access of the enzyme to its substrate, which results in the release of free [3H]N-acetylneuraminic acid. This release was detected through the transfer of radioactivity from a trichloroacetic acid-insoluble to a trichloroacetic acid-soluble fraction. An ATP-dependent component of release was found, which appears to be a consequence of vesicle fusion. This component was enhanced when the donor was prepared from cells in which the enzyme had been concentrated in a compartment between the Golgi complex and the plasma membrane, which indicates that a specific exocytic fusion event has been reconstituted. The extent of fusion is greatly reduced by pre-treatment of donor and acceptor preparations with trypsin, which points to the involvement of proteins in the fusion reaction.

scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

scholarly journals The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin.

1986 ◽  
Vol 34 (8) ◽  
pp. 1069-1077 ◽  
Author(s):  
K Hedman ◽  
I Pastan ◽  
M C Willingham
Keyword(s):  
Plasma Membrane ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Coated Pits ◽  
Golgi Stack ◽  
Acetylneuraminic Acid ◽  
Secretory Glycoproteins ◽  
Double Label ◽  
Endocytic Vesicles ◽  
Exocytic Pathway

The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.

scholarly journals TCR–pMHC bond conformation controls TCR ligand discrimination

2019 ◽  
Vol 17 (3) ◽  
pp. 203-217 ◽  
Author(s):  
Dibyendu K. Sasmal ◽  
Wei Feng ◽  
Sobhan Roy ◽  
Peter Leung ◽  
Yanran He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Feedback Loop ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsic Property ◽  
Unanswered Question ◽  
Structural Differences ◽  
Self Peptides

Abstract A major unanswered question is how a TCR discriminates between foreign and self-peptides presented on the APC surface. Here, we used in situ fluorescence resonance energy transfer (FRET) to measure the distances of single TCR–pMHC bonds and the conformations of individual TCR–CD3ζ receptors at the membranes of live primary T cells. We found that a TCR discriminates between closely related peptides by forming single TCR–pMHC bonds with different conformations, and the most potent pMHC forms the shortest bond. The bond conformation is an intrinsic property that is independent of the binding affinity and kinetics, TCR microcluster formation, and CD4 binding. The bond conformation dictates the degree of CD3ζ dissociation from the inner leaflet of the plasma membrane via a positive calcium signaling feedback loop to precisely control the accessibility of CD3ζ ITAMs for phosphorylation. Our data revealed the mechanism by which a TCR deciphers the structural differences among peptides via the TCR–pMHC bond conformation.

In situ measurement of plasma membrane hemileaflet susceptibility to peroxidation

1993 ◽  
Vol 15 (5) ◽  
pp. 539
Author(s):  
D. Jourd'Heuil ◽  
S. Mehta ◽  
J.B. Meddings
Keyword(s):  
Plasma Membrane ◽  
In Situ Measurement

scholarly journals Dating minerals by ID-TIMS geochronology at times of in situ analysis: selected case studies from the CPGeo-IGc-USP laboratory

2009 ◽  
Vol 81 (1) ◽  
pp. 73-97 ◽  
Author(s):  
Cláudia R. Passarelli ◽  
Miguel A.S. Basei ◽  
Oswaldo Siga Jr. ◽  
Kei Sato ◽  
Walter M. Sproesser ◽  
...  
Keyword(s):  
Thermal Ionization Mass Spectrometry ◽  
Age Determination ◽  
Isotopic Dilution ◽  
Ionization Mass ◽  
System Behavior ◽  
Rock Types ◽  
Geological Processes ◽  
Thermal Ionization Mass

Since 1964, the Center for Geochronological Research - CPGeo, one of the interdepartmental centers of the Instituto de Geociências (IG) of São Paulo University, has developed studies related to several geological processes associated with different rock types. Thermal Ionization Mass Spectrometry Isotopic Dilution (ID-TIMS) has been the technique widely used in the CPGeo U-Pb Laboratory. It provides reliable and accurate results in age determination of superposed events. However, the open-system behavior such as Pb-loss, the inheritance problem and metamictization processes allow and impel us to a much richer understanding of the power and limitations of U-Pb geochronology and thermochronology. In this article, we present the current methodology used at the CPGeo-IGc-USP U-Pb laboratory, the improvements on ID-TIMS method, and report high-precision U-Pb data from zircon, monazite, epidote, titanite, baddeleyite and rutile from different rock types of several domains of the Brazilian south-southeast area, Argentina and Uruguay.

scholarly journals Freeze-stable sialidase activity in human leucocytes: substrate specificity, inhibitor susceptibility, detergent requirements and subcellular localization

1994 ◽  
Vol 301 (3) ◽  
pp. 777-784 ◽  
Author(s):  
P J Waters ◽  
A P Corfield ◽  
R Eisenthal ◽  
C A Pennock
Keyword(s):  
Plasma Membrane ◽  
Heparan Sulphate ◽  
Subcellular Fractionation ◽  
Sodium Cholate ◽  
Acidic Ph ◽  
Sialidase Activity ◽  
Acetylneuraminic Acid ◽  
Mononuclear Leucocytes ◽  
Radioactive Assay ◽  
Assay Conditions

Human leucocytes contain a freeze-stable sialidase (neuraminidase; EC 3.2.1.18) activity in addition to the better-characterized lysosomal freeze-labile enzyme. In order to discriminate between the sialidase activities detected with the synthetic fluorimetric substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (MU-Neu5Ac), different tritiated sialoglycoconjugate substrates were prepared. Using this sensitive radioactive assay system, leucocyte sialidase activity towards glycoproteins was shown to be labile to repeated freeze-thawing, but a Triton-stimulated activity towards gangliosides was entirely freeze-stable. Assay conditions were optimized for this freeze-stable ganglioside sialidase activity. Subcellular fractionation of mononuclear leucocytes (MNLs) on Percoll-density gradients showed that this ganglioside sialidase activity was entirely associated with the plasma membrane. Study of the detergent requirements showed that MNLs also demonstrated ganglioside sialidase activity when sodium cholate was present in place of Triton. Cholate-stimulated ganglioside sialidase activity was found to be entirely freeze-stable and localized at the plasma membrane. Studies on whole homogenates of MNLs demonstrated that the Triton-stimulated and cholate-stimulated activities showed similar acidic pH optima at < or = 3.9 and were both strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and Cu2+, but not by free N-acetylneuraminic acid, N-(4-nitrophenyl)oxamic acid or heparan sulphate. These results suggest that human MNLs contain, in addition to the lysosomal freeze-labile sialidase, a single sialidase activity which is freeze-stable, ganglioside-specific, plasma membrane-associated and stimulated both by Triton and by cholate.

scholarly journals Isolation and characterization of the urothelial lumenal plasma membrane.

1977 ◽  
Vol 73 (2) ◽  
pp. 382-399 ◽  
Author(s):  
J S Caruthers ◽  
M A Bonneville
Keyword(s):  
Plasma Membrane ◽  
Sodium Dodecyl ◽  
Modified Technique ◽  
Sialic Acid Content ◽  
Polarity Index ◽  
Total Sialic Acid ◽  
Isolation And Characterization ◽  
A Value

The lumenal plasma membrane has been isolated from transitional epithelial cells (urothelium) lining the urinary bladder in sheep by a modified technique involving treatment with hypotonic thioglycolate. The isolated membranes, like those in situ, are distinguished morphologically by arrays of hexagonal particles (in plague regions) separated by smooth interplaque regions. These plaque regions, specifically, can be isolated from the lumenal plasma membrane. Of the proteins constituting the lumenal plasma membrane, five were found to characterize the plaque regions and, in particular, the 33,000-dalton species appears to be most heavily concentrated in the sodium dodecyl sulfate-polyacrylamide gel pattern of the isolated plaque regions. Lipid analyses showed that there are approximately 0.93 mg of phospholipid and 0.27 mg of cholesterol for each milligram of protein, giving a value of 55% lipids and 45% proteins for the composition of the lumenal plasma membrane. The total sialic acid content was measured to be approximately 0.038 micronmol/mg protein for the plasma membrane. Several plasma membrane marker enzymes were found to be associated with the lumenal plasma membrane fraction, but only the 5'-nucleotidase activity was found to be further enriched in the plaque region fraction. Amino acid analysis of the intrinsic proteins of the plaques indicated a polarity index of 45%.

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