scholarly journals Studies on cell adhesion and recognition. III. The occurrence of α-mannosidase at the fibroblast cell surface, and its possible role in cell recognition

1981 ◽  
Vol 88 (1) ◽  
pp. 149-159 ◽  
Author(s):  
H Rauvala ◽  
S Hakomori
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Inhibitory Activity ◽  
Time Course ◽  
Gas Chromatography Mass Spectrometry ◽  
Hydrolytic Cleavage ◽  
Intact Cells ◽  
Cell Monolayers ◽  
Mannose 6 Phosphate ◽  
Hydrolysis Of

The occurrence of α-mannosidase activity at the surface of hamster embryo (NIL) fibroblasts is indicated by the following findings: (a) When NIL cells were incubated on the glass surfaces on which ovalbumin glycopeptides were covalently linked, a rapid release of free mannose from ovalbumin glycopeptides was observed as evidenced by analysis on gas chromatography/mass spectrometry. (b) Cell suspensions as well as intact cell monolayers hydrolyzed rapidly p-nitrophenyl-α-D-mannoside, and the time-course of the hydrolytic cleavage was linear from the moment of mixing of the substrate with the cells. The hydrolysis of the nitrophenyl glycosides of β-D-mannose, α-D-galactose, β-D-galactose, α-L-fucose, β-D-glucose, β-D-N-acetylgalactosamine and β-D-N-acetylglucosamine was negligible or more than ten times lower as compared with the hydolysis of α-D-mannoside. (c) No released or secreted activity of mannosidase could be detected under the conditions used. (d) Studies using known proportions of broken cells in the incubation mixture indicated that more than 90 percent of the mannosidase activity measured was attributable to intact cells and not to broken cells or cell fragments. (e) Hydrolysis of p-nitrophenyl-α-D-mannoside by cell monolayers was inhibited, in the order of decreasing inhibitory activity, by yeast mannan, ovalbumin, α-1,4-L-mannonolactone, α-methylmannoside, and mannose-6-phosphate. High inhibitory activity of the mannan polysaccharide and of ovalbumin favored the presence of the mannosidase activity at the cell surface, as these substrates may not penetrate rapidly into the cells. The following findings indicated that the cell surface mannosidase is mediating the cell adhesion based on the recognition of high-mannose-type glycopeptide: (a) Ovalbumin- coated plastic surfaces strongly promoted attachment and spreading of NIL fibroblasts, whereas the same ovalbumin coat did not promote attachment and spreading of some other cell types (BALB/c 3T3 fibroblasts and freshly prepared rat liver cells). (b) Digestion of ovalbumin with α-mannosidase greatly reduced the adhesion-mediating activity. (c) Cell adhesion to ovalbumin-coated surfaces was strongly inhibited by mannose tetrasaccharides, moderately by α-1,4-L-mannonolactone, and weakly by α- methylmannoside and mannose-6-phosphate. This order of the inhibitory activity for cell attachment is the same as that for the inhibition of mannosidic hydrolysis. The interpretation that the cell surface mannosidase is able to mediate cell adhesion is in agreement with previous studies suggesting that polyvalent glycosidase surfaces can promote cell adhesion to a degree similar to that caused by fibronectin and several lectins by interacting with their cell surface substrate site (the accompanying papers of this series).

Basolateral sorting signal of the 300-kDa mannose 6-phosphate receptor

2002 ◽  
Vol 282 (1) ◽  
pp. G51-G60 ◽  
Author(s):  
Debra A. Wick ◽  
Bellur Seetharam ◽  
Nancy M. Dahms
Keyword(s):  
Cell Surface ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Endosomal Trafficking ◽  
Surface Distribution ◽  
Cell Monolayers ◽  
Cytoplasmic Region ◽  
Mannose 6 Phosphate ◽  
Mutant Receptors ◽  
Golgi Network

In polarized cells, the delivery of numerous membrane proteins from the trans-Golgi network to the basolateral surface depends on specific sequences located in their cytoplasmic domain. We have previously shown that the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) exhibits a polarized cell surface distribution in the human colon adenocarcinoma (Caco-2) cell line in which there is a threefold enrichment on the basolateral surface. To investigate the role of residues in the cytoplasmic region of the receptor that facilitates its entry into the basolateral sorting pathway, we generated stably transfected Caco-2 cell lines expressing various mutant bovine IGF-II/MPRs. The steady-state surface distribution of mutant receptors was analyzed by subjecting filter-grown cell monolayers to incubation with iodinated IGF-II/MPR-specific antibody or to indirect immunofluorescence and visualization by confocal microscopy. Together, these results demonstrate that the sorting of the IGF-II/MPR to the basolateral cell surface depends on recognition of sequences located in its cytoplasmic region that are distinct from the Tyr-based internalization and dileucine-dependent endosomal trafficking motifs.

scholarly journals Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes

1996 ◽  
Vol 315 (2) ◽  
pp. 635-641 ◽  
Author(s):  
Louise E. DONNELLY ◽  
Nigel B. RENDELL ◽  
Stephen MURRAY ◽  
Jennifer R. ALLPORT ◽  
Gar LO ◽  
...  
Keyword(s):  
Cell Surface ◽  
Time Course ◽  
Surface Proteins ◽  
Polymorphonuclear Neutrophil ◽  
Transferase Activity ◽  
Nad Glycohydrolase ◽  
Snake Venom Phosphodiesterase ◽  
Molecular Masses ◽  
Human Polymorphonuclear Neutrophil ◽  
Hydrolysis Of

An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1–10 mM) did not yield ADP-ribosyl-DEA-BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD+ by an NAD+ glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The Km of the enzyme for NAD+ was 100.1±30.4 μM and the Vmax 1.4±0.2 pmol of ADP-ribosylagmatine/h per 106 cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98% decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [32P]NAD+. Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5´-AMP.

scholarly journals Stimulation of Na+ transport by AVP is independent of PKA phosphorylation of the Na-K-ATPase in collecting duct principal cells

2005 ◽  
Vol 289 (5) ◽  
pp. F1031-F1039 ◽  
Author(s):  
David Mordasini ◽  
Mauro Bustamante ◽  
Martine Rousselot ◽  
Pierre-Yves Martin ◽  
Udo Hasler ◽  
...  
Keyword(s):  
Cell Surface ◽  
Time Course ◽  
Collecting Duct ◽  
Pump Current ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intact Cells ◽  
Principal Cells ◽  
Na Pump ◽  
Stimulation Of

Arginine-vasopressin (AVP) stimulates Na+ transport and Na-K-ATPase activity via cAMP-dependent PKA activation in the renal cortical collecting duct (CCD). We investigated the role of the Na-K-ATPase in the AVP-induced stimulation of transepithelial Na+ transport using the mpkCCDc14 cell model of mammalian collecting duct principal cells. AVP (10−9 M) stimulated both the amiloride-sensitive transepithelial Na+ transport measured in intact cells and the maximal Na pump current measured by the ouabain-sensitive short-circuit current in apically permeabilized cells. These effects were associated with increased Na-K-ATPase cell surface expression, measured by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. The effects of AVP on Na pump current and Na-K-ATPase cell surface expression were dependent on PKA activity but independent of increased apical Na+ entry. Time course experiments revealed that in response to AVP, the cell surface expression of both endogenous Na-K-ATPase and hybrid Na pumps containing a c- myc-tagged wild-type human α1-subunit increased transiently. Na-K-ATPase cell surface expression was maximal after 30 min and then declined toward baseline after 60 min. Immunoprecipitation experiments showed that PKA activation did not alter total phosphorylation levels of the endogenous Na-K-ATPase α-subunit. In addition, mutation of the PKA phosphorylation site (S943A or S943D) did not alter the time course of increased cell surface expression of c- myc-tagged Na-K-ATPase in response to AVP or to dibutyryl-cAMP. Therefore, stimulation of Na-K-ATPase cell surface expression by AVP is dependent on PKA but does not rely on α1-subunit phosphorylation on serine 943 in the collecting duct principal cells.

scholarly journals Binding, interiorization and degradation of cholesterol ester-labelled chylomicron-remnant particles by rat hepatocyte monolayers

1977 ◽  
Vol 168 (3) ◽  
pp. 483-494 ◽  
Author(s):  
Claes-Henrik Florén ◽  
Åke Nilsson
Keyword(s):  
Cell Surface ◽  
Cholesteryl Ester ◽  
Time Course ◽  
Inhibitory Effect ◽  
Metabolic Inhibitors ◽  
High Density Lipoproteins ◽  
Chylomicron Remnant ◽  
Very Low Density Lipoproteins ◽  
Chylomicron Remnants ◽  
Hydrolysis Of

1. The cholesteryl ester of isolated chylomicron-remnant particles was efficiently degraded by hepatocyte monolayers. The degradation was sensitive to metabolic inhibitors. 2. With increasing amounts of remnant cholesteryl ester the rate of uptake approached saturation and conformed to a linear double-reciprocal plot. The Vmax. was determined as 80ng of cholesteryl ester/h per mg of protein and the apparent Km as 1.4μg of cholesteryl ester per mg of protein. The time course for the uptake and hydrolysis suggested that binding of particles to the cell surface preceded the degradation. 3. Cholesteryl esters of native chylomicrons were degraded to a much smaller extent and their presence had only a small inhibitory effect on the degradation of chylomicron remnants. Intestinal very-low-density lipoproteins were degraded somewhat faster than chylomicrons, and caused more inhibition of remnant degradation. Rat high-density lipoproteins inhibited the hydrolysis of remnant cholesteryl ester by up to 50%, but had less influence on the amount of cholesteryl ester that was bound to the cells. Serum decreased both the uptake and hydrolysis, whereas d=1.21 infranatant had no effect. 4. The cholesteryl ester hydrolysis after the uptake by the cells was inhibited by chloroquine and by colchicine. Only 28–36% of the unhydrolysed cholesteryl ester could be released from these cells by trypsin treatment, indicating that the major portion was truly intracellular. The particles that could be released from the cell surface by trypsin and those remaining in the medium had the same triacylglycerol/cholesteryl ester ratio as the added remnant particles. Significant amounts of denser particles were thus not formed during contact with the cell surface. 5. The presence of heparin, as well as preincubation of the cells with heparin, increased the uptake of chylomicron remnants. This effect was most marked in the presence of serum. A much smaller proportion of the other serum lipoproteins was taken up, and this proportion was not increased by heparin.

Mechanism of diminished contractile response to catecholamines during acidosis

1988 ◽  
Vol 254 (1) ◽  
pp. H20-H27 ◽  
Author(s):  
J. D. Marsh ◽  
T. I. Margolis ◽  
D. Kim
Keyword(s):  
Cell Surface ◽  
Intracellular Ph ◽  
Adrenergic Receptor ◽  
Time Course ◽  
Contractile Response ◽  
Beta Adrenergic Receptor ◽  
Similar Time ◽  
Intact Cells ◽  
Beta Adrenergic ◽  
Surface Receptors

To examine mechanisms of diminished contractile response to catecholamines during acidosis, we studied contractile properties, beta-adrenergic receptor properties, and intracellular pH of intact, cultured myocardial cells from chick embryo ventricle at pH 7.4 and 6.8. Contractile response was measured with an optical-video system. On changing the superfusing buffer from pH 7.4 to 6.8 there was a decline in contractile amplitude to 80% of control by 20 min. Fluorimetrically determined intracellular pH declined over a similar time course from 7.11 +/- 0.05 to 6.96 +/- 0.07 (P less than 0.05). After 45 min at pH 6.8 the contractile response to 1 microM isoproterenol was less than half of the response at pH 7.4. Antagonist and agonist ligand-binding properties of the beta-adrenergic receptor were determined in the intact cells under conditions identical to those for the contractility studies. With the use of the hydrophilic antagonist [3H]CGP-12177 that selectively labels cell-surface receptors, agonist competition studies demonstrated that acidosis had no significant effect on antagonist or agonist affinity but decreased beta-receptor number from 21 +/- 3 to 11 +/- 3 fmol/mg protein (P less than 0.02). It is probable that a decline in the number of beta-receptors on the cell surface contributes to contractile hyporesponsiveness to catecholamines during acidosis.

scholarly journals The time course of hydrolysis of a β-lactam antibiotic by intact gram-negative bacteria possessing a periplasmic β-lactamase

1987 ◽  
Vol 244 (3) ◽  
pp. 509-513 ◽  
Author(s):  
W W Nichols
Keyword(s):  
Time Course ◽  
Decay Constant ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Beta Lactam ◽  
Intact Cells ◽  
Gram Negative ◽  
First Order ◽  
Lactam Antibiotic ◽  
Hydrolysis Of

An equation is derived from first principles for describing the change in concentration with time of a beta-lactam antibiotic in the presence of intact Gram-negative bacteria possessing a beta-lactamase located in the periplasmic space. The equation predicts a first-order decline in beta-lactam concentration of the form [S] = [Si]e lambda t, where [S] is the exogenous concentration of beta-lactam, [Si] is the value of [S] at time zero, t is the time from mixing of cells and antibiotic and lambda (less than 0) is the decay constant. The value of lambda is exactly described by the theory in terms of experimentally measurable quantities. Quantitative data concerning cephaloridine hydrolysis by intact cells of Haemophilus influenzae agreed well with the theory, as did data concerning the uptake of 2-nitrophenyl galactoside by intact cells of Escherichia coli. Cephalosporin C hydrolysis by intact cells of Pseudomonas aeruginosa did not progress as predicted by the theory. The theory is applicable to any substrate which is acted on by an enzyme that is located solely in the periplasmic space and that obeys the Michaelis-Menten equation of enzyme kinetics.

Oncornavirus assembly at the cell surface revealed in replicas of freeze-dried cells

1978 ◽  
Vol 36 (2) ◽  
pp. 354-355
Author(s):  
Anthony Demsey ◽  
Christopher W. Stackpole
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Viral Origin ◽  
Intact Cells ◽  
Structural Formation ◽  
Virus Core ◽  
Freeze Dried ◽  
Cacodylate Buffer ◽  
Surface Replica ◽  
Leukemia Viruses

The murine leukemia viruses are type-C oncornaviruses, and their release from the host cell involves a “budding” process in which the newly-forming, RNA-containing virus core becomes enveloped by modified cell surface membrane. Previous studies revealed that the released virions possess a dense array of 10 nm globular projections (“knobs”) on this envelope surface, and that these knobs contain a 70, 000 MW glycoprotein (gp70) of viral origin. Taking advantage of this distinctive structural formation, we have developed a procedure for freeze-drying and replication of intact cells which reveals surface detail superior to other surface replica techniques, and sufficient to detect even early stages of virus budding by localized aggregation of these knobs on the cell surface.Briefly, cells growing in monolayer are seeded onto round glass coverslips 10-12 mm in diameter. After a period of growth, cells are fixed in situ for one hour, usually with 1% OsO4 in 0. 1 M cacodylate buffer, and rinsed in distilled water.

A pre-embedding, protein a-gold immunolabelling technique for cytocentrifuged cell monolayers for lm and tem

1986 ◽  
Vol 44 ◽  
pp. 220-221
Author(s):  
K. Chien ◽  
I.P. Shintaku ◽  
A.F. Sassoon ◽  
R.L. Van de Velde ◽  
R. Heusser
Keyword(s):  
Cell Surface ◽  
Staining Method ◽  
Protein A ◽  
Surface Antigens ◽  
Cell Suspensions ◽  
Cellular Morphology ◽  
Cellular Phenotype ◽  
Cell Surface Antigens ◽  
Cell Monolayers ◽  
Diagnostic Histopathology

Identification of cellular phenotype by cell surface antigens in conjunction with ultrastructural analysis of cellular morphology can be a useful tool in the study of biologic processes as well as in diagnostic histopathology. In this abstract, we describe a simple pre-embedding, protein A-gold staining method which is designed for cell suspensions combining the handling convenience of slide-mounted cell monolayers and the ability to evaluate specimen staining specificity prior to EM embedding.

Determination of δ-hexadecansultone in sodium α-olefinesulphonates and liquid detergents using gas chromatography-mass spectrometry (GC/MS)

2019 ◽  
Vol 85 (7) ◽  
pp. 16-21
Author(s):  
Liliya R. Mubarakova ◽  
German K. Budnikov
Keyword(s):  
Multicomponent Mixture ◽  
Operating Mode ◽  
Complete Separation ◽  
Gas Chromatography Mass Spectrometry ◽  
Liquid Detergent ◽  
Cyclic Esters ◽  
Alcohol Water ◽  
Household Chemicals ◽  
Hydrolysis Of

Sultones are cyclic esters of hydroxysulfonic acids, which are formed in the process of sulfonation of α-olefins with sulfur trioxide gas. More stable sultones may be present in the final product — an anionic surfactant — sodium α-olefin sulfonate (AOC-Na). AOC-Na is widely used in the production of household chemicals and cosmetic products, including liquid dishwashing detergents. Sultones are strong skin sensitizers, their level in AOC-Na should be strictly controlled and not exceed 5 ppm. Operational and strict control of the sultone content upon AOC-Na production allows timely adjustment at the stage of hydrolysis, which leads to a more complete disclosure of the sultone cycle with the formation of the corresponding olefin sulfonates and hydroxyalkanesulfonates. We propose a method for determining δ-hexadecansultone in liquid dishwashing detergents and sodium α-olefinsulfonates obtained on the basis of α-olefins of C14 – C16 fractions using GC/MS, which provides shortening of sample preparation and keeps the sensitivity with a detection limit of 0.02 mg/kg. The effect of various weakly polar and non-polar organic solvents used for Sultone extraction from AOC-Na and liquid detergent on liquid extraction based on the dispersion of the extractant in an alcohol/water phase is studied. When selecting the solvent we have shown that the use of diethyl ether provided the best extraction of the analyte. Determination of the analyte extraction recovery was performed using the reaction of hydrolysis of the extracted mixture. We specified the operating mode of the device which provided complete separation of the components of the analyzed compounds including the samples of liquid detergent for dishes being a multicomponent mixture of complex composition.

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

1985 ◽  
Vol 108 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Nandalal Bagchi ◽  
Birdie Shivers ◽  
Thomas R. Brown
Keyword(s):  
Time Course ◽  
Period 2 ◽  
Iodotyrosine Deiodinase ◽  
Rate Of Hydrolysis ◽  
Underlying Mechanisms ◽  
Excess Iodide ◽  
Sites Of Action ◽  
Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

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