Coating Of Beads Research Articles

Cytoskeletal mechanics in adherent human airway smooth muscle cells: probe specificity and scaling of protein-protein dynamics.

We probed elastic and loss moduli in the adherent human airway smooth muscle cell through a variety of receptor systems, each serving as a different molecular window on cytoskeletal dynamics. Coated magnetic microbeads were attached to the cell surface via coating-receptor binding. A panel of bead coatings was investigated: a peptide containing the sequence RGD, vitronectin, urokinase, activating antibody against beta(1)-integrin, nonactivating antibody against beta(1)-integrin, blocking antibody against beta(1)-integrin, antibody against beta(1)-integrin, and acetylated low-density lipoprotein. An oscillatory mechanical torque was applied to the bead, and resulting lateral displacements were measured at baseline, after actin disruption by cytochalasin D, or after contractile activation by histamine. As expected, mechanical moduli depended strongly on bead type and bead coating, differing at the extremes by as much as two orders of magnitude. In every case, however, elastic and loss moduli increased with frequency f as a weak power law, f( x-1). Moreover, with few exceptions, data could be scaled such that elastic and frictional responses depended solely on the power law exponent x. Taken together, these data suggest that power law behavior represents a generic feature of underlying protein-protein dynamics.

Immulectin-2, a pattern recognition receptor that stimulates hemocyte encapsulation and melanization in the tobacco hornworm, Manduca sexta

In insects, encapsulation followed by melanization is a major defense mechanism against metazoan parasites. However, insects must recognize and differentiate nonself before they mount an immune response. Recognition of pathogens in insects is accomplished by a set of pattern recognition receptors (PRRs). Binding of PRRs to pathogens is linked to a variety of immune responses including phagocytosis, nodule formation, encapsulation, and prophenoloxidase activation. So far, little is known about how recognition of pathogens by PRRs triggers different immune responses. In this article, we report that immulectin-2, a C-type lectin, enhances encapsulation and melanization processes in the tobacco hornworm, Manduca sexta. Coating of agarose beads with recombinant carboxyl-terminal carbohydrate-recognition domain (CRD2-II) of immulectin-2 enhanced encapsulation of the beads in vitro by hemocytes and melanization of the beads in vivo in M. sexta larvae. Recombinant CRD2-II also directly bound to granular cells and oenocytoids, but not to plasmatocytes or spherule cells. Immulectin-2 in hemolymph of M. sexta larvae bound to the surface of a nematode, Caenorhabditis elegans, and recombinant CRD2-II directly bound to C. elegans and a human filarial nematode, Brugia malayi. Binding of CRD2-II to C. elegans enhanced melanization of the nematode in vivo. Our results suggest that binding of immulectin-2 to the surface of parasites can trigger encapsulation and melanization responses in M. sexta.

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3′(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.

Cell Release from Alginate Immobilized Lactococcus lactis ssp. lactis in Chitosan and Alginate Coated Beads

The effects of chitosan and alginate coatings of alginate beads with entrapped Lactococcus lactis ssp. lactis were studied in batch and continuous fermentations. Chitosan coating reduced the final concentrations of free cells, the initial release of free cells and the rate of lactate production in milk fermented batch-wise to a final pH of 4.7 in five consecutive batch fermentations. An alternative experimental system based on continuous fermentation with controlled pH and a high dilution rate was developed to better study the phenomenon of cell release. To estimate the effects of different bead coatings on cell release, alginate beads were coated with chitosan or alginate, or sequentially with chitosan/alginate or chitosan/alginate/chitosan. Chitosan coating alone seemed to reduce the rate of cell release only in the early stages of the fermentation, while sequential coatings with chitosan and alginate showed significant reduction throughout the whole test period. To examine whether the observed effects of bead coating could be explained only by a decrease in cell activity, the ratios between the rate of cell release and the rate of lactate production were examined during the fermentations for the different beads. This ratio showed qualitatively the same behavior as direct results of volumetric cell release.

Immunomagnetic separation of cells of the toxic dinoflagellate Alexandrium fundyense from natural plankton samples

A novel method was developed for isolating cells of the toxic dinoflagellate Alexandrium fundyense from preserved natural seawater samples using paramagnebc beads and a monoclonal antibody against the surface antigens of the dinoflagellate. 'Direct' and 'indirect' approaches to bead/cell attachment were tested as well as 3 types of bead coatings (streptavidin, and 2 secondary antibodies: sheep anti-mouse, and goat anti-mouse), and 2 blocking agents [normal goat serum (NGS) and bovine serum albumin (BSA)]. Optimal 'indirect' bead attachment protocols, where the species-specific pnmary antibody is first bound to the target cells before bead attachment, utilized either 2.8 pm streptavidin-coated beads with NGS blocking or sheep anti-mouse-coated beads with BSA blocking. Although there were undoubtably some cell losses during the initial antibody-labeling and washing procedures, ca 90% of the labeled A. fundyense cells in unialgal cultures were removed using these bead procedures. Non-specific binding was low, as only 5 to 10 % of the A. fundyense cells were recovered when the primary antibody was omitted. The 'direct' approach, where the species-specific primary antibody is first bound to the beads, was most effective (80% recovery) using sheep anti-mousecoated beads (2.8 p) wlth BSA blocking, and the non-specific binding remained very low ( ~ 2 % ) . When tested in 3 natural preserved plankton samples, recovery of Alexandrium spp. was ca 90% (negative controls <10%) using the 'indirect' method, with 5% contamination by other species. The 'direct' technique was slightly less effective on the natural samples (ca 80% recovery), but negative controls were very low at 1% or less. Contam~nation by other species was 5 to 10%. 'Direct' attachment is a simpler procedure than the 'indirect' approach because the beads can be pre-coated with the speciflc antibody in bulk before use. Thus, the direct method not only shortens the procedure by eliminating the need to antibody-label each individual sample but, as a consequence, minimizes target cell losses as well, so it may be the method of choice for working with field samples. Immunomagnetic separation of a slngle algal species from a sample containing mixed plankton and detritus IS thus simple, rapid, and quite reliable. Compared with other sorting methods currently in use (e.g. manual pipette isolation, flow cytometry), this procedure offers distinct advantages with respect to cost, volume, speed and simplicity.

Bead Coating Process Via an Excess of Crosslinking Agent

AbstractCoated beads were prepared by soaking in sodium alginate solutions spherical matrices (beads) of carboxymethylcellulose crosslinked with aluminum chloride (AlCl3) and loaded with ambroxol hydrochloride as a model drug. The residual amount of the crosslinker induced an interfacial crosslinking reaction of the sodium alginate. Therefore, an insoluble, smooth and uniform in thickness coat was formed around the beads. As the coating time increased, the coat thickness increased until1 AlCl3 was present inside the beads. The rate of drug release from the coated beads was slower than that from the uncoated beads and decreased with the increase in coating time. Moreover, a constant rate phase, subsequent a burst period for the samples obtained with the highest coating times, was achieved. The dynamic swelling analysis allowed to exclude the influence of the polymer relaxation on the release process which appeared to be controlled by the alginate coat.

Bead Coating: II. Effect of Spheronization Technique on Drug Release from Coated Spheres

AbstractThe effect of spheronization method on drug release from coated spheres may be evaluated by determining the drug release rate, the critical coating level and the release mechanism. Drug release is faster from pan beads than from marumerizer beads at the same coating level. An equation is proposed which indicates that the critical coating level is inversely proportional to sphere size and sphere density, which in turn results from the different spheronization techniques. From the calculation, the critical coating levels for 14/16 mesh cuts of marumerizer beads and pan beads are 12% and 18%, respectively. Disintegration, pore-control and barrier control are involved in the release mechanisms of drugs from coated pan beads.

Phagocytic interactions of sialated glycoprotein, sugar, and lectin coated beads with rat retinal pigment epithelium.

Latex beads coated with mucin (a sialic acid containing glycoconjugate), N-acetylglucosamine (GlcNAc), or with the lectins, succinylated wheat germ agglutinin (sWGA) (binds to GlcNAc) or Limax flavus agglutinin (LFA) (binds to sialic acid residues) were used as phagocytic particles. Phagocytosis of these coated beads by retinal pigment epithelial (RPE) explants was determined by bead uptake in normal (Long Evans) and dystrophic (Royal College of Surgeons, RCS/p+) rat retinas. Electron microscopy showed that beads coated with mucin or LFA lectin were not phagocytized by either dystrophic or normal RPE. sWGA-coated beads were phagocytized by both dystrophic and normal RPE, while GlcNAc-coated beads were taken up by dystrophic RPE only. Specificity of uptake for sWGA and GlcNAc bead coatings was shown by the reduction in the number of beads phagocytized in the presence of the appropriate competing sugar or lectin. The lack of phagocytic uptake of beads coated with a sialated glycoprotein or a sialic acid binding lectin suggests that sialic acid residues are not recognized as particulate ligands in this phagocytic assay. The data which show the uptake of beads coated with sWGA (binds only GlcNAc) together with results which showed WGA (binds both GlcNAc and sialic acid)-coated beads were not taken up, further suggest that GlcNAc residues may be involved in bead phagocytosis. The most striking difference between normal and dystrophic RPE engulfment of coated beads is the uptake of GlcNAc-coated beads by dystrophic RPE only. These results suggest that the receptor molecules on dystrophic RPE cell surface membranes may be different from those on normal RPE membranes.