scholarly journals Endocytosis of hyaluronan in rat Kupffer cells

1992 ◽  
Vol 286 (2) ◽  
pp. 519-526 ◽  
Author(s):  
J Alston-Smith ◽  
H Pertoft ◽  
T C Laurent
Keyword(s):  
Kupffer Cells ◽  
Culture Medium ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Fluid Phase ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Intracellular Degradation ◽  
Acetyl Group ◽  
A Minor

The binding, uptake and degradation of hyaluronan (HA) labelled with 3H in its acetyl group were studied in cultured rat Kupffer cells (KC). At 4 degrees C the binding increased with increasing concentrations of HA in the culture medium up to at least 1 microgram/ml, when saturation occurred. Binding could be prevented efficiently by the addition of an excess of unlabelled HA, and to a lesser extent by chondroitin sulphate and oligosaccharide fragments of HA, consisting of four sugars or more. The labelled HA bound to the cells could be removed by incubating the cells with Streptomyces hyaluronidase, or trypsin, indicating that the HA-binding sites are located on the cell surface. At 37 degrees C HA was internalized in a concentration-dependent manner, and degradation products appeared in the supernatant after 1-5 h, depending on the concentration applied. At 50 ng of free HA/ml, each KC accumulated 60 ag of the polysaccharide/min in the first 1 h, and degraded a total amount of 10 fg of HA during an 8 h period. Addition of the negatively charged polysaccharide dextran sulphate reduced binding, and to an even greater extent internalization, of HA in KC, while no effect was observed with dextran. Depletion of intracellular potassium caused a marked reduction in the rate of endocytosis of cell-membrane-associated HA into KC, without affecting binding. Addition of KCl to the culture medium returned endocytosis of [3H]HA to normal levels. There was no effect on binding and a partial effect on internalization by depletion of bivalent cations or in the presence of EDTA. The degradation of [3H]HA by KC cultures was abolished in the presence of weak bases, NH4Cl and chloroquine, supporting the idea that HA is endocytosed into lysosomes prior to degradation. The fluid-phase marker [14C]sucrose was internalized in the cells at much lower rate than was HA. Rates of binding, internalization and degradation of HA in KC point therefore to a specific endocytosis followed by an intracellular degradation to low-M(r) compounds. It was estimated that, under physiological conditions, KC only clear a minor proportion of circulating HA.

The Role of the αC Domains in the Formation of Nonadhesive Fibrinogen Matrices

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1194-1194
Author(s):  
Ivan S Yermolenko ◽  
Fuhrmann Alexander ◽  
Oleg V Gorkun ◽  
Valeryi K Lishko ◽  
Susan T Lord ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Degradation Products ◽  
Structural Basis ◽  
Dependent Manner ◽  
Adhesion Forces ◽  
Concentration Dependent Manner ◽  
Matrix Interactions ◽  
The Matrix

Abstract Abstract 1194 Fibrinogen strongly reduces adhesion of platelets and leukocytes to the surface of fibrin clots, highlighting a possible role for this abundant plasma protein in surface-mediated control of thrombus growth, stability and timely dissolution. We have previously shown that the underlying mechanism by which fibrinogen adsorprtion on various surfaces decreases cell adhesion is its aggregation, leading to the formation of an extensible multilayered matrix. This matrix is incapable of transducing strong mechanical forces via integrins resulting in insufficient intracellular signaling and weak cell adhesion. To further characterize the properties of the nonadhesive fibrinogen multilayer and determine the structural basis for its formation, we compared the physical and adhesive properties of fibrinogen matrices prepared from human plasma fibrinogen (hFg), recombinant normal fibrinogen (rFg) and fibrinogen with the truncated αC domains (FgAα251). Using atomic force microscopy (AFM) and force spectroscopy we determined the thickness, adhesion forces, extensibility and the energy of the AFM tip-fibrinogen matrix interactions of various matrices. All three fibrinogens adsorbed on mica in a concentration-dependent manner. However, while hFg and rFg formed the matrix with a maximal thickness of ∼8 nm corresponding to 8–9 molecular layers, the deposition of FgAα251 was terminated after 2–3 layers, indicating the αC domains are involved in the formation of the multilayer. Consistently, the extensibility of the full multilayered matrix prepared from hFg and rFg was 2-fold higher than that of the matrix formed from FgAα251 (57±6 nm and 28±4 nm for hFg and FgAα251, respectively). The formation of the multilayered matrix upon adsorption of the increasing concentrations of hFg and FgAα251 was associated with a decrease in the adhesion forces generated between the AFM tip and the matrix. However, the energy required to disrupt the AFM tip-matrix interactions was 1.6 nN·nm for hFg and 0.88 nN·nm for FgAα251, indicating that the αC domains contribute about half of the total energy to the fibrinogen-fibrinogen interactions during the formation of the multilayer. Since cell adhesion inversely correlates with the growth of the fibrinogen matrices, we examined adhesion of U937 monocytic cells to the substrates prepared from different concentrations of hFg and FgAα251. As expected, adhesion sharply declined with the increase in the coating concentration of hFg, with only 2–5% of the cells adhering to the full multilayer. At the same time, 40–45% of the cells remained adherent on the matrices prepared from FgAα251. These results indicate that the inability of FgAα251 to assemble a highly extensible multilayer correlates with enhanced cell adhesion. The increased adhesiveness of matrices formed from fibrinogen with truncated αC domains may have implications for situations where the formation of fibrinogen degradation products with truncated αC domains such as X-fragment occurs, for example during thrombolytic therapy and pathological fibrinogenolysis, as well as in the cases of disfibrinogenemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen

1984 ◽  
Vol 223 (2) ◽  
pp. 401-412 ◽  
Author(s):  
R M Mason ◽  
J D Lineham ◽  
M A Phillipson ◽  
C M Black
Keyword(s):  
Culture Medium ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Primary Cultures ◽  
Normal Subjects ◽  
Dermal Fibroblasts ◽  
Proteoglycan Synthesis ◽  
Dependent Manner ◽  
Glycoprotein Synthesis ◽  
Chondrocyte Cultures

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.

scholarly journals Impact of Natural IgM Concentration on Gene Therapy with Adenovirus Type 5 Vectors

2014 ◽  
Vol 89 (6) ◽  
pp. 3412-3416 ◽  
Author(s):  
Qi Qiu ◽  
Zhili Xu ◽  
Jie Tian ◽  
Rituparna Moitra ◽  
Sreenivasulu Gunti ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Kupffer Cells ◽  
Adenovirus Type ◽  
Dependent Manner ◽  
Lower Efficiency ◽  
Concentration Dependent Manner ◽  
Natural Igm ◽  
Adenovirus Type 5

Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentrationin vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice.

scholarly journals Catabolism of aggrecan, decorin and biglycan in tendon

2000 ◽  
Vol 350 (1) ◽  
pp. 181-188 ◽  
Author(s):  
Sarah G. REES ◽  
Carl R. FLANNERY ◽  
Chris B. LITTLE ◽  
Clare E. HUGHES ◽  
Bruce CATERSON ◽  
...  
Keyword(s):  
Culture Medium ◽  
Core Protein ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Expression Patterns ◽  
Collagen Matrix ◽  
Pcr Analysis ◽  
Age Related ◽  
Proteoglycan Degradation

We have examined the catabolism of the proteoglycans aggrecan, decorin and biglycan in fresh tendon samples and in explant cultures of tissue from the tensional and compressed regions of young and mature bovine tendons. A panel of well-characterized antibodies that recognize glycosaminoglycan or protein (linear or neoepitope) sequences was used to detect proteoglycans and proteoglycan degradation products that were both retained within the tissue and released into the culture medium. In addition, a reverse-transcriptase-mediated PCR analysis was used to examine the mRNA expression patterns of tendon proteoglycans and aggrecanases. The results of this study indicate a major role for aggrecanase(s) in the catabolism of aggrecan in bovine tendon. The study also provides a characterization of glycosaminoglycan epitopes associated with the proteoglycans of tendon, illustrating age-related changes in the isomers of chondroitin sulphate disaccharides that remain attached to the core protein glycosaminoglycan linkage region after digestion with chondroitinase ABC. Evidence for a rapid turnover of the small proteoglycans decorin and biglycan was also observed, indicating additional molecular pathways that might compromise the integrity of the collagen matrix and potentially contribute to tendon dysfunction after injury and during disease.

scholarly journals Chondroitin Sulfate Induces Depression of Synaptic Transmission and Modulation of Neuronal Plasticity in Rat Hippocampal Slices

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Elisa Albiñana ◽  
Javier Gutierrez-Luengo ◽  
Natalia Hernández-Juarez ◽  
Andrés M. Baraibar ◽  
Eulalia Montell ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Synaptic Transmission ◽  
Neuronal Plasticity ◽  
Chondroitin Sulphate ◽  
Hippocampal Slices ◽  
Long Term Potentiation ◽  
Dependent Manner ◽  
Perineuronal Net ◽  
Concentration Dependent Manner ◽  
Population Spike Amplitude

It is currently known that in CNS the extracellular matrix is involved in synaptic stabilization and limitation of synaptic plasticity. However, it has been reported that the treatment with chondroitinase following injury allows the formation of new synapses and increased plasticity and functional recovery. So, we hypothesize that some components of extracellular matrix may modulate synaptic transmission. To test this hypothesis we evaluated the effects of chondroitin sulphate (CS) on excitatory synaptic transmission, cellular excitability, and neuronal plasticity using extracellular recordings in the CA1 area of rat hippocampal slices. CS caused a reversible depression of evoked field excitatory postsynaptic potentials in a concentration-dependent manner. CS also reduced the population spike amplitude evoked after orthodromic stimulation but not when the population spikes were antidromically evoked; in this last case a potentiation was observed. CS also enhanced paired-pulse facilitation and long-term potentiation. Our study provides evidence that CS, a major component of the brain perineuronal net and extracellular matrix, has a function beyond the structural one, namely, the modulation of synaptic transmission and neuronal plasticity in the hippocampus.

Phosphatidylcholine participates in the interaction between macrophages and lymphocytes

2000 ◽  
Vol 278 (3) ◽  
pp. C554-C560 ◽  
Author(s):  
Anita Nishiyama-Naruke ◽  
Rui Curi
Keyword(s):  
Arachidonic Acid ◽  
Transfer Process ◽  
Concanavalin A ◽  
Molecular Species ◽  
Lymphocyte Proliferation ◽  
Culture Medium ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Physiological Importance

The role of phosphatidylcholine molecules as mediator for the control of lymphocyte proliferation by macrophages was investigated. Phosphatidylcholine added to the culture medium inhibited the concanavalin A-stimulated lymphocyte proliferation in a concentration-dependent manner. The potency of this effect was dependent on the presence of arachidonic acid in the phosphatidylcholine molecules. The phosphatidylcholine transfer from macrophages to lymphocytes was then investigated. Macrophages incorporated phosphatidylcholine at a much higher rate than lymphocytes and exported phosphatidylcholine to the culture medium. When cocultured, a significant amount of phosphatidylcholine incorporated by macrophages was transferred to lymphocytes. To examine the possible physiological importance of the transfer process, the lymphocyte proliferation was measured in coculture conditions. Macrophages were treated with phosphatidylcholine and washed, and then these cells were cocultured with concanavalin A-stimulated lymphocytes. The effect observed in coculture was an inhibition of lymphocyte proliferation, which was also dependent on the molecular species of the phosphatidylcholine. Therefore, phosphatidylcholine may act as a mediator of the macrophage effect on lymphocyte proliferation.

scholarly journals Migration by haptotaxis of a Schwann cell tumor line to the basement membrane glycoprotein laminin.

1983 ◽  
Vol 97 (3) ◽  
pp. 772-777 ◽  
Author(s):  
J B McCarthy ◽  
S L Palm ◽  
L T Furcht
Keyword(s):  
Cell Movement ◽  
Fluid Phase ◽  
Basement Membranes ◽  
Boyden Chamber ◽  
Dependent Manner ◽  
Binding Studies ◽  
Directed Movement ◽  
Concentration Dependent Manner ◽  
Filter Surface

Laminin is a large (greater than 850-kdalton) glycoprotein that is localized within basement membranes. Recent work has indicated that this protein is present within the endoneurium of mouse sciatic nerve. Furthermore, it has been shown that a rat Schwannoma cell line, RN22F, produced laminin and that laminin promoted the attachment of these cells to bacterial plastic. This report presents evidence that RN22F cells migrate in vitro to laminin in a concentration-dependent fashion. Laminin was extracted from the mouse EHS tumor and purified by molecular sieve and heparin-agarose affinity chromatography. The migration of Schwannoma cells to laminin, as assessed in a microwell modified Boyden chamber, was inhibited in a dose-dependent manner by affinity-purified antilaminin antibody. Zigmond-Hirsch checkerboard analysis experiments indicated that laminin stimulated both random and directed movement of RN22F cells. Additionally, reversal of the laminin gradient in the chambers also stimulated RN22F migration in a concentration-dependent manner, suggesting that directed migration of RN22F cells was due to a substratum-bound laminin (haptotaxis) as opposed to cell movement in response to fluid-phase laminin (chemotaxis). Binding studies using [3H]laminin demonstrated that laminin bound to the filter surface under the assay conditions used, and support the contention that cells are migrating to substrate-bound material. Furthermore, RN22F cells were shown to migrate on filters coated with laminin in the absence of additional fluid-phase laminin. The magnitude of this response could be altered by changing the relative density of bound laminin. In contrast, fibronectin promoted only marginal migration of RN22F cells. Collectively, these observations indicate that haptotaxis may be a mechanism by which laminin may guide cells during development and raise the possibility that it may be involved in peripheral nervous system myelination.

Natriuretic peptides inhibit mesangial cell production of endothelin induced by arginine vasopressin

1993 ◽  
Vol 264 (4) ◽  
pp. F678-F683 ◽  
Author(s):  
M. Kohno ◽  
T. Horio ◽  
M. Ikeda ◽  
K. Yokokawa ◽  
T. Fukui ◽  
...  
Keyword(s):  
Arginine Vasopressin ◽  
Natriuretic Peptides ◽  
High Performance ◽  
Culture Medium ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells ◽  
Concentration Dependent Manner ◽  
Medium Level

The present study examined the effects of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively) on endothelin-1 (ET-1) secretion after stimulation with arginine vasopressin (AVP), using cultured rat glomerular mesangial cells. AVP stimulated immunoreactive (ir) ET-1 secretion in a concentration-dependent manner via a receptor-mediated process. Rat ANP-(1-28) and rat BNP-45 potently inhibited this stimulated secretion in a concentration-dependent manner. Inhibition by ANP and BNP of AVP-stimulated ET-1 secretion was paralleled by an increase in the medium level of guanosine 3',5'-cyclic monophosphate (cGMP). The addition of a cGMP analogue, 8-bromo-cGMP, reduced the stimulated ET-1 secretion. CNP was much less effective than rat ANP-(1-28) or rat BNP-45 with respect to inhibiting irET-1 secretion and increasing cGMP levels. High-performance liquid chromatography indicated that the major component of irET-1 in the culture medium corresponds to ET-1-(1-21). These findings indicate that AVP stimulates ET-1 secretion in cultured rat mesangial cells and that rat ANP and BNP inhibit this stimulated secretion, probably through a cGMP-dependent process.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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