concanavalin-a and hydroxyethyl-methacrylate

In this study, the objective was to separate exopolysaccharides (EPSs) released in the broth subsequent to outdoor cultivation of Botryococcus braunii. For this, poly(2-hydroxyethyl methacrylate) (PHEMA) cryogels were synthesized. After that, the surface was modified by coupling Concanavalin A. Box-Behnken statistical design was used to evaluate the effect of freezing temperature, Con A concentration and flow rate on Con A binding capacity. Optimum synthesis conditions were elicited as -14.48°C freezing temperature, 1.00mg/ml Con A concentration and 0.30ml/min flow rate yielding 3.18mg Con A/g cryogel, whereas -16°C, 1.00mg/ml and 0.30ml/min yielded the highest (3.38mg) binding capacity in experimental cryogel preparation. The EPS adsorption capacity of the optimum cryogel column was found as 3.26mg EPS/g cryogel corresponding to adsorption yield of 80%. Besides; swelling test, elemental analysis, Micro-CT, SEM and FTIR analysis were carried out for characterization of the synthesized cryogels.

Topics: Chemistry Techniques, Synthetic; Chlorophyta; Concanavalin A; Cryogels; Hydrophobic and Hydrophilic Interactions; Methacrylates; Polymerization; Polysaccharides; Surface Properties; Temperature

In this work we have developed a hydrophilic poly(hydroxyethyl methacrylate-co-poly(ethylene glycol) diacrylate) cryogel placed in the centrifugal filter device. The composition of the polymerization mixture as well as the polymerization conditions were optimized in order to prepare a material with bimodal pore size distribution with 20-50 μm flow through macropores and submicrometer pores in the polymer walls. The optimized, mechanically stable, highly porous, material was used for spin column lectin chromatography. The surface of the monolithic scaffold was activated by epichlorohydrin and used for immobilization of concanavalin A to provide the affinity supports for selective isolation of glycoproteins containing high mannose glycan structures. The performance of the developed lectin modified cryogels was evaluated by analyses of glycoprotein mixtures. The efficiency and selectivity of the affinity supports were confirmed by MALDI-MS analysis.

Topics: Chromatography, Liquid; Concanavalin A; Cryogels; Glycoproteins; Methacrylates; Polyethylene Glycols; Polymerization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The unique characteristics of polypropylene non-woven meshes (PPNWMs), like random network of overlapped fibers, multiple connected pores and overall high porosity, make them high potentials for use as separation or adsorption media. Meanwhile, carbohydrates can specifically recognize certain lectin through multivalent interactions. Therefore glycosylated PPNWMs, combing the merits of both, can be regarded as superior affinity membranes for lectin adsorption and purification. Here, we describe a versatile strategy for the glycosylation of PPNWMs. Two hydrophilic polymers with different side chain length, poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(oligo(ethylene glycol) methacrylate) (POEGMA), were first conformally tethered on the polypropylene fiber surface by a modified plasma pretreatment and benzophenone (BP) entrapment UV irradiation process. Then glucose ligands were bound through the reaction between the hydroxyl group and acetyl glucose. Chemical changes of the PPNWMs surface were monitored by FT-IR/ATR. SEM pictures show that conformal glucose ligands can be achieved through the modified process. After deprotection, the glycosylated PPNWMs became superhydrophilic and had high specific recognition capability toward Concanavalin A (Con A). Static Con A adsorption experiments were further performed and the results indicate that fast adsorption kinetics and high binding capacity can be accomplished at the same time. We also found that increasing the side chain length of polymer brushes had positive effect on protein binding capacity due to improved chain mobility. Model studies suggest a multilayer adsorption behavior of Con A.

Topics: Adsorption; Benzophenones; Chromatography, Affinity; Concanavalin A; Fluorescence; Glucose; Glycosylation; Kinetics; Ligands; Methacrylates; Plasma Gases; Polyethylene Glycols; Polymethacrylic Acids; Polypropylenes; Spectroscopy, Fourier Transform Infrared; Time Factors; Ultraviolet Rays

A glycomonomer was synthesized from poly(ethylene glycol) methacrylate (PEGMA). The terminal hydroxyl moieties were activated with ester groups and subsequently the glucosamine was incorporated forming urethane linkages. The obtained glycomonomer was copolymerized with methyl acrylate by free radical polymerization varying the initial feed composition to produce different amphiphilic glycopolymers. The glycopolymers were then characterized and compared with the homologous glycopolymers based on 2-{[(D-glucosamin-2-N-yl)carbonyl]oxy}ethyl methacrylate. Both series of glycopolymers were used in emulsion polymerization of methyl acrylate as stabilizers without the addition of any cosurfactant. Although high conversions were not achieved with any of the employed surfactant, the glycopolymers provide good colloidal stability, spherical, monodisperse and small latex particles in comparison with the surfactant-free emulsion polymerization. The latex particles stabilized with the glycosurfactant based on PEGMA, containing a flexible spacer between the backbone and the glucosamine, lead to smooth films whereas the short side chain surfactant from 2-hydroxyethyl methacrylate (HEMA), with higher glass transition temperature, restricts the coalescence of particles and, therefore, the film formation. Moreover, the surface bioactivity of these polymer coatings was examined by analyzing their specific interaction with the lectin, Concanavalin A, Canavalia ensiformis. The specific and successful binding to the Concanavalin A was demonstrated by fluorescence microscopy for both series being more intense with increasing amount of glycounits in the glycopolymer stabilizers. Interestingly, the incorporation of a flexible spacer in the glycopolymer structures enhances the binding activity.

Topics: Acrylates; Colloids; Concanavalin A; Emulsions; Glucosamine; Latex; Methacrylates; Microscopy, Fluorescence; Particle Size; Polyethylene Glycols; Polymerization; Protein Binding; Surface Properties; Surface-Active Agents

Individuals working in a dental clinic are exposed to 2-hydroxyethyl methacrylate (HEMA). HEMA has been found to have several effects on the immune system, including acting as an adjuvant in mice and stimulating the production of human IgG1 in vitro. In this study we continued to explore the immunomodulatory properties of HEMA in mice. Mice were co-injected subcutaneously with the following: HEMA + ovalbumin (OVA) in bicarbonate buffer, OVA in bicarbonate buffer, HEMA in bicarbonate buffer, or bicarbonate buffer alone. Mice immunized with OVA were killed 2 wk after a booster injection. Mice exposed to HEMA only were killed 6 d after the last injection with HEMA. Serum and spleens were collected. The activities of anti-OVA IgG and anti-OVA IgM were determined using ELISAs, as was the in vitro production of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by splenocytes after 2 d of incubation. Splenocyte proliferation was analyzed using [(3) H]thymidine decomposition. Mice exposed twice to HEMA in vivo had a higher baseline and a higher concanavalin A-stimulated proliferation of splenocytes, and produced less TNF-α in relation to IL-6, compared with controls. Immunization of mice with OVA/HEMA resulted in a higher anti-OVA IgG activity, relative to anti-OVA IgM activity, compared with controls. In conclusion, HEMA has selective effects on cytokine and antibody production in mice.

Topics: Animals; Bicarbonates; Buffers; Cell Proliferation; Concanavalin A; Dental Materials; Immunization; Immunoglobulin G; Immunoglobulin M; Immunologic Factors; Injections, Subcutaneous; Interleukin-6; Male; Methacrylates; Mice; Mice, Inbred BALB C; Mitogens; Ovalbumin; Specific Pathogen-Free Organisms; Spleen; Tumor Necrosis Factor-alpha

An improved postembedding method for electron microscopic localization of concanavalin A (Con A) receptors in ultrathin sections is reported. Materials are embedded in a hydrophilic resin, glycol methacrylate copolymerized with glutaraldehyde and urea, using a precisely controlled polymerization schedule that preserves ultrastructural integrity. Ferritin-Con A binds specifically in ultrathin sections of mouse spleen tissue and of Sephadex beads embedded in this resin. Quantitative investigations of procedures for decreasing nonspecific labeling demonstrate that preincubation of sections with bovine serum albumin reduces background to a very low level. This high-resolution method allows macromolecular affinity probes access to receptors in all cellular locations, while maintaining good morphological preservation.

Topics: Animals; Concanavalin A; Cytoplasm; Endoplasmic Reticulum; Female; Ferritins; Freezing; Glutaral; Histocytochemistry; Lymphocytes; Methacrylates; Mice; Mice, Inbred A; Microscopy, Electron; Receptors, Concanavalin A; Serum Albumin, Bovine; Spleen