The Characterisation and Quantification of Immobilised Concanavalin A on Quartz Surfaces Based on The Competitive Binding to Glucose and Fluorescent Labelled Dextran

The competition between various carbohydrates in the binding to Concanavalin A (Con A) can be exploited in gravimetric microsensors that detect changes in mass or viscoelasticity as a function of glucose concentration. Such sensors are based on the immobilisation of Con A as the ligand specific element, and a successful application requires that the binding property of Con A is retained. This paper presents a simplified immobilisation procedure of Con A on a quartz surface, a common material for gravimetric microsensors. Structural assessment with atomic force microscopy confirmed that the surface was covered with a layer of macromolecules. This layer shows the presence of entities of various sizes, presumably monomers, dimers and tetramers among which dimers of the Con A are the most dominant structure. Functional assessment using fluorescent labelled dextran (FITC and Alexa 488) suggests a surface coverage ranging from 1.8 × 1011 to 2.1 × 1012 immobilised fluorescent molecules per cm2. The assay was responsive to glucose over a concentration range from 0–40 mM, but became gradually saturated above 20 mM. Hence, the immobilised Con A is able to bind dextran, which is displaced by glucose in a concentration dependent manner, thus triggering a mass change proportional to the MW of dextran.

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A C21-Steroidal Glycoside from Cynanchum atratum Attenuates Concanavalin A-Induced Liver Injury in Mice

Molecules ◽

10.3390/molecules24061087 ◽

2019 ◽

Vol 24 (6) ◽

pp. 1087 ◽

Cited By ~ 2

Author(s):

Jian Yang ◽

Bin Wang ◽

Chao-feng Zhang ◽

Xiang-hong Xu ◽

Mian Zhang

Keyword(s):

T Lymphocytes ◽

Concanavalin A ◽

Underlying Mechanism ◽

Dependent Manner ◽

Histopathological Changes ◽

Con A ◽

Concentration Dependent Manner ◽

Steroidal Glycoside ◽

First Time

Cynatratoside A (CyA) is a C21 Steroidal glycoside with pregnane skeleton isolated from the root of Cynanchum atratum Bunge (Asclepiadaceae). This study aimed to investigate the effects of CyA on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) and the underlying mechanism. CyA was orally administered to mice at 10 and 40 mg/kg 8 h before and 1 h after Con A treatment. The effects of CyA on Con A-induced spleen and liver in mice were assessed via histopathological changes, T lymphocyte amounts and the expressions of IL-1β and ICAM-1. Con A-induced L-02 hepatocytes were used to evaluate whether CyA (0.1–10 μM) can directly protect hepatocytes from cytotoxicity and the possible mechanism. The results revealed that CyA treatment could significantly improve the histopathological changes of spleen and liver, reduce the proliferation of splenic T lymphocytes, and decrease the expressions of IL-1β and ICAM-1 in liver. The experiment in vitro showed that CyA inhibited Con A-induced hepatotoxicity in a concentration-dependent manner. CyA (10 μM) significantly increased/decreased the expression of Bcl-2/Bax and reduced the levels of cleaved caspases-9 and -3. Our study demonstrated for the first time that CyA has a significant protective effect on Con A-induced AIH by inhibiting the activation and adhesion of T lymphocytes and blocking hepatocyte apoptosis.

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Probing Changes in Ca2+-Induced Interaction Forces between Calmodulin and Melittin by Atomic Force Microscopy

Micromachines ◽

10.3390/mi11100906 ◽

2020 ◽

Vol 11 (10) ◽

pp. 906

Author(s):

Sheng Huang ◽

Jianhua Wang ◽

Heng Sun ◽

Yuna Fu ◽

Yan Wang

Keyword(s):

Atomic Force Microscopy ◽

Biological Macromolecules ◽

Dependent Manner ◽

Adhesion Forces ◽

Concentration Dependent Manner ◽

Force Microscopy ◽

Calmodulin Binding ◽

Atomic Force ◽

Relationship Of ◽

Configuration Changes

Mechanobiology studies the means by which physical forces and mechanical properties change intra- or inter- biological macromolecules. Calmodulin (CaM) is involved in physiological activities and various metabolic processes in eukaryotic cells. Although the configuration changes in the interaction between calmodulin and melittin have been studied, the biomechanical relationship of their interaction has rarely been explored. Here, we measured the adhesion forces between calmodulin and melittin in solutions of gradient concentration of calcium ions using atomic force microscopy (AFM). We found that the specific (Fi) and nonspecific (F0) adhesion forces between single melittin and calmodulin in a PBS solution were 69.4 ± 5.0 and 29.3 ± 8.9 pN, respectively. In the presence of 10−7 to 10−3 M Ca2+ PBS solution, the Fi increased significantly to 93.8 ± 5.0, 139.9 ± 9.0, 140.4 ± 9.7, 171.5 ± 9.0, and 213.3 ± 17.8 pN, indicating that the unbinding force between melittin and calmodulin increased in the presence of Ca2+ in a concentration-dependent manner. These findings demonstrated that biomechanical studies based on AFM could help us better understand the melittin/calmodulin-binding processes in the presence of calcium and help us design and screen peptide drugs based on calmodulin.

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Atomic force microscopy measurement of leukocyte-endothelial interaction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00491.2003 ◽

2004 ◽

Vol 286 (1) ◽

pp. H359-H367 ◽

Cited By ~ 80

Author(s):

Xiaohui Zhang ◽

Aileen Chen ◽

Dina De Leon ◽

Hong Li ◽

Eisei Noiri ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Inflammatory Diseases ◽

Leukocyte Adhesion ◽

Umbilical Vein ◽

Quantitative Measure ◽

Dependent Manner ◽

Average Force ◽

Force Microscopy ◽

Atomic Force ◽

Umbilical Vein Endothelial Cells

Leukocyte adhesion to vascular endothelium is a key initiating step in the pathogenesis of many inflammatory diseases. In this study, we present real-time force measurements of the interaction between monocytic human promyelocytic leukemia cells (HL-60) cells and a monolayer of human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy (AFM). The detachment of HL-60-HUVEC conjugates involved a series of rupture events with force transitions of 40–100 pN. The integrated force of these rupture events provided a quantitative measure of the adhesion strength on a whole cell level. The AFM measurements revealed that HL-60 adhesion is heightened in the borders formed by adjacent HUVECs. The average force and mechanical work required to detach a single HL-60 from the borders of a tumor necrosis factor-α-activated HUVEC layer were twice as high as those of the HUVEC bodies. HL-60 adhesion to the monolayer was significantly reduced by a monoclonal antibody against β1-integrins and partially inhibited by antibodies against selectins ICAM-1 and VCAM-1 but was not affected by anti-αVβ3. Interestingly, adhesion was also inhibited in a dose-dependent manner (IC50≈ 100 nM) by a cyclic arginine-glycine-aspartic acid (cRGD) peptide. This effect was mediated via interfering with the VLA-4-VCAM-1 binding. In parallel measurements, transmigration of HL-60 cells across a confluent HUVEC monolayer was inhibited by the cRGD peptide and by both anti-β1and anti-αVβ3antibodies. In conclusion, these data demonstrate the role played by β1-integrins in leukocyte-endothelial adhesion and transmigration and the role played by αVβ3in transmigration, thus underscoring the high efficacy of cRGD peptide in blocking both the adhesion and transmigration of monocytes.

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Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton

Biochemical Journal ◽

10.1042/bj2410521 ◽

1987 ◽

Vol 241 (2) ◽

pp. 521-525 ◽

Cited By ~ 7

Author(s):

S M Gokhale ◽

N G Mehta

Keyword(s):

Concanavalin A ◽

Membrane Skeleton ◽

Cross Linking ◽

Dependent Manner ◽

Con A ◽

Normal Cells ◽

Skeletal Protein ◽

Low Ionic Strength ◽

Dose Dependent ◽

Resistance To Heat

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.

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Modulation of human lymphocyte function by C3a and C3a(70-77).

Journal of Experimental Medicine ◽

10.1084/jem.156.3.756 ◽

1982 ◽

Vol 156 (3) ◽

pp. 756-765 ◽

Cited By ~ 27

Author(s):

D G Payan ◽

D E Trentham ◽

E J Goetzl

Keyword(s):

T Lymphocytes ◽

Mononuclear Cells ◽

Interaction Analysis ◽

Direct Interaction ◽

Mononuclear Leukocytes ◽

Lymphocyte Function ◽

Dependent Manner ◽

Con A ◽

Concentration Dependent Manner ◽

Human Mononuclear Cells

Human C3a and the synthetic octapeptide C3a (70-77), which retains the activities of an anaphylatoxin, inhibit in a concentration-dependent manner the generation of leukocyte inhibitory factor (LIF) activity by human mononuclear leukocytes and T lymphocytes cultured with the mitogens phytohemagglutinin (PHA) or concanavalin A (Con A) or the antigen streptokinase-streptodornase (SK-SD). The generation of LIF activity was inhibited by 50% by 10(-8) M C3a or C3a(70-77) with PHA or Con A as the stimulus, whereas a more than 10-fold higher concentration of C3a(70-77) than C3a was required to achieve the same level of suppression with SK-SD as the stimulus. Similar concentrations of C3a(70-77) inhibited to the same extent the migration of T lymphocytes stimulated by alpha-thioglycerol of Con A. Neither C3a nor C3a(70-77) altered significantly the uptake of [3H]thymidine by human mononuclear cells exposed to PHA, Con A, or SK-SD. The capacity of C3a(70-77)-Sepharose,m but not Sepharose alone, to adsorb or inactivate mononuclear leukocytes required for the generation of LIF activity established a direct interaction. Analysis of the lymphocytes in the effluent from C3a(70-77)-Sepharose columns, using monoclonal antibodies to surface antigens, showed a selective depletion of the helper/inducer population of lymphocytes. C3a might represent an important mediator of the functionally selective regulation of human T lymphocyte activities by the complement system.

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Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton.

The Journal of Cell Biology ◽

10.1083/jcb.92.2.565 ◽

1982 ◽

Vol 92 (2) ◽

pp. 565-573 ◽

Cited By ~ 67

Author(s):

R G Painter ◽

M Ginsberg

Keyword(s):

Concanavalin A ◽

Surface Membrane ◽

Dnase I ◽

Actin Binding ◽

Resting Cells ◽

Dependent Manner ◽

Con A ◽

Intact Cells ◽

Platelet Surface ◽

Surface Glycoproteins

We have measured the association of platelet surface membrane proteins with Triton X-100 (Triton)-insoluble residues in platelets surface labeled with 125I. In both concanavalin A (Con A)-stimulated and resting platelets, this fraction is composed largely of polypeptides with apparent molecular weights of 45,000, 200,000, and 250,000 which comigrate with authentic actin, myosin heavy chain, and actin binding protein, respectively, as judged by PAGE in SDS. Less than 10% of the two major 125I-labeled surface glycoproteins, GPiib and GPIII, were associated with the Triton residue in resting platelets. Within 45 s after Con A addition, 80-95% of these two glycoproteins became associated with the Triton residue and the amount of sedimentable actin doubled. No cosedimentation of GPIIb and III with the cytoskeletal protein-containing Triton residue was seen when Con A was added to a Triton extract of resting cells, indicating that the sedimentation of GPIIb and III seen in Con A-stimulated platelets was not due to precipitation of the glycoproteins by Con A after detergent lysis. Treatment of Triton extracts of Con A-stimulated platelets with DNase I (deoxyribonucleate 5'-oligonucleotidido-hydrolase [EC 3.1.4.5]) inhibited the sedimentation of actin and the two surface glycoproteins in a dose-dependent manner. This inhibition of cosedimentation was not due to an effect of DNase I on Con A-glycoprotein interactions since these two glycoproteins could be quantitatively recovered by Con A-Sepharose affinity absorption in the presence of DNase I. When the Con A bound to the Triton residue was localized ultrastructurally, it was associated with cell-sized structures containing filamentous material. In intact cells, there was simultaneous immunofluorescent coredistribution of surface-bound Con A and myosin under conditions which induced a redistribution of platelet myosin. These data suggest that Con A can, in the intact platelet, induce physical interactions between certain surface glycoproteins and the internal cytoskeleton.

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Fish-Derived Antifreeze Proteins and Antifreeze Glycoprotein Exhibit a Different Ice-Binding Property with Increasing Concentration

Biomolecules ◽

10.3390/biom10030423 ◽

2020 ◽

Vol 10 (3) ◽

pp. 423 ◽

Cited By ~ 1

Author(s):

Sakae Tsuda ◽

Akari Yamauchi ◽

N. M.-Mofiz Uddin Khan ◽

Tatsuya Arai ◽

Sheikh Mahatabuddin ◽

...

Keyword(s):

Concentration Dependence ◽

Thermal Hysteresis ◽

Antifreeze Proteins ◽

Biochemical Properties ◽

Binding Property ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ice Binding ◽

Binding Function ◽

The Difference

The concentration of a protein is highly related to its biochemical properties, and is a key determinant for its biotechnological applications. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are structurally diverse macromolecules that are capable of binding to embryonic ice crystals below 0 °C, making them useful as protectants of ice-block formation. In this study, we examined the maximal solubility of native AFP I–III and AFGP with distilled water, and evaluated concentration dependence of their ice-binding property. Approximately 400 mg/mL (AFP I), 200 mg/mL (AFP II), 100 mg/mL (AFP III), and >1800 mg/mL (AFGP) of the maximal solubility were estimated, and among them AFGP’s solubility is much higher compared with that of ordinary proteins, such as serum albumin (~500 mg/mL). The samples also exhibited unexpectedly high thermal hysteresis values (2–3 °C) at 50–200 mg/mL. Furthermore, the analysis of fluorescence-based ice plane affinity showed that AFP II binds to multiple ice planes in a concentration-dependent manner, for which an oligomerization mechanism was hypothesized. The difference of concentration dependence between AFPs and AFGPs may provide a new clue to help us understand the ice-binding function of these proteins.

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Influence of Nano, Micro and Macro Topography of Dental Implant Surfaces on Human Gingival Fibroblasts

International Journal of Molecular Sciences ◽

10.3390/ijms22189871 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9871

Author(s):

Morena Petrini ◽

Tania Vanessa Pierfelice ◽

Emira D’Amico ◽

Natalia Di Pietro ◽

Assunta Pandolfi ◽

...

Keyword(s):

Soft Tissues ◽

Titanium Implant ◽

Sem Analysis ◽

Human Gingival Fibroblasts ◽

Dependent Manner ◽

Gingival Fibroblasts ◽

Force Microscopy ◽

Atomic Force

Current research on dental implants has mainly focused on the influence of surface roughness on the rate of osseointegration, while studies on the development of surfaces to also improve the interaction of peri-implant soft tissues are lacking. To this end, the first purpose of this study was to evaluate the response of human gingival fibroblasts (hGDFs) to titanium implant discs (Implacil De Bortoli, Brazil) having different micro and nano-topography: machined (Ti-M) versus sandblasted/double-etched (Ti-S). The secondary aim was to investigate the effect of the macrogeometry of the discs on cells: linear-like (Ti-L) versus wave-like (Ti-W) surfaces. The atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis showed that the Ti-S surfaces were characterized by a significantly higher micro and nano roughness and showed the 3D macrotopography of Ti-L and Ti-W surfaces. For in vitro analyses, the hGDFs were seeded into titanium discs and analyzed at 1, 3, and 5 days for adhesion and morphology (SEM) viability and proliferation (Cck-8 and MTT assays). The results showed that all tested surfaces were not cytotoxic for the hGDFs, rather the nano-micro and macro topography favored their proliferation in a time-dependent manner. Especially, at 3 and 5 days, the number of cells on Ti-L was higher than on other surfaces, including Ti-W surfaces. In conclusion, although further studies are needed, our in vitro data proved that the use of implant discs with Ti-S surfaces promotes the adhesion and proliferation of gingival fibroblasts, suggesting their use for in vivo applications.

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Phosphatidylcholine participates in the interaction between macrophages and lymphocytes

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.3.c554 ◽

2000 ◽

Vol 278 (3) ◽

pp. C554-C560 ◽

Cited By ~ 18

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.

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