Binding of wheat germ agglutinin to extracellular network produced by cultured human fibroblasts

The occurrence of diverse carbohydrate moieties on the cell surface and in the extracellular matrix, makes lectins the suitable probes to study the distribution of appropriate determinants produced in cell culture. Biotin-labelled wheat germ agglutinin (WGA) was used in microscopic and photometric detection of lectin binding to monolayer of human skin fibroblasts. The incubation of confluent fibroblast monolayer with labelled WGA reveals two principal patterns of binding of this lectin: to cell surface structures and, predominantly, to extracellular fibres; the alignment and density of extracellular network are not uniform. After binding of WGA to confluent culture, light microscopic analysis revealed the ubiquitous fibrillar network between and over cells, with some regions of increased compactness and altered orientation of fibrils. Binding to cell surfaces (manifested as specks) was predominant for the fibroblasts at the logarithmic phase of growth. N-acetylglucosamine (0.2 M) and native lectin (100 microg/ml) had a partial inhibitory effect on WGA binding to the extracellular network. Treatment with neuraminidase (0.1 unit/ml) of untreated or prefixed monolayers resulted in a significant decrease in WGA binding to fibrils (and increase in PNA binding), indicating that terminal sialic acid residues are mainly involved in the network-WGA interaction. Mild trypsinization (10 microg/ml) removed the target sites, which retained the ability to bind WGA, being spotted on hydrophobic Immobilon P paper; biotinylated lectin, bound to adsorbed glycopeptides, could be eluted and quantified in solid-phase inhibition assay.     

Effects of Carbon Nanotubes in a Chitosan/Collagen-Based Composite on Mouse Fibroblast Cell Proliferation

This study investigated the in vitro cytocompatibility of carbon nanotubes (CNTs) in a chitosan/collagen-based composite. Mouse fibroblasts were cultured on the surface of a novel material consisting of CNTs in a chitosan/collagen-based composite (chitosan/collagen+CNTs group). Chitosan/collagen composites without CNTs served as the control material (chitosan/collagen group) and cells cultured normally in tissue culture plates served as blank controls (blank control group). Cell adhesion and proliferation were observed, and cell apoptosis was measured. The doubling time (DT₁) of cells was significantly shorter in the chitosan/collagen+CNTs group than in the chitosan/collagen group, and that in the chitosan/collagen group was shorter than in the blank control group. The CNTs in the chitosan/collagen-based composites promoted mouse fibroblast adhesion, producing a distinct cytoskeletal structure. At 24 h after culture, the cytoskeleton of the cells in the chitosan/collagen+CNTs group displayed typical fibroblastic morphology, with clear microfilaments. Cells in the chitosan/collagen group were typically round, with an unclear cytoskeleton. The blank control group even had a few unattached cells. At 4 days after incubation, no early apoptosis of cells was detected in the blank control group, whereas early apoptosis of cells was observed in the chitosan/collagen+CNTs and chitosan/collagen groups. No significant difference in the proportion of living cells was detected among the three groups. After entering the plateau stage, the average cell number in the chitosan/collagen+CNTs group was similar to that in the chitosan/collagen group and significantly smaller than that in the blank control group. Early apoptosis of cells in the blank control group was not detectable. There were significant differences in early apoptosis among the three groups. These results suggest that CNTs in a chitosan/collagen-based composite did not cause significant cytotoxic effects on mouse fibroblasts. Compared with chitosan/collagen composites, early adhesion and proliferation of fibroblasts were increased on chitosan/collagen+CNTs. However, at relatively high cell densities, the CNTs in the chitosan/collagen-based composite might exert an inhibitory effect on mouse fibroblast proliferation by inducing apoptosis.     

Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts

Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and MHR-alpha), carrot (MHR-C) and potato (MHR-P) cells. Polystyrene (PS) Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of the MHRs. Results clearly demonstrate that MHR-B induces cell adhesion, proliferation and survival, in contrast to the other MHRs. Moreover, MHR-alpha causes cells to aggregate, decrease proliferation and enter into apoptosis. Cells cultured in standard conditions with 1% soluble MHR-B or MHR-alpha show the opposite behaviour to the one observed on MHR-B and -alpha-grafted PS. Fibronectin was similarly adsorbed onto MHR-B and tissue culture polystyrene (TCPS) control, but poorly on MHR-alpha. The Fn cell binding site (RGD sequence) was more accessible on MHR-B than on TCPS control, but poorly on MHR-alpha. The disintegrin echistatin inhibited fibroblast adhesion and spreading on MHR-B-grafted PS, which suggests that MHRs control fibroblast behaviour via serum-adhesive proteins. This study provides a basis for the design of intelligently-tailored biomaterial coatings able to induce specific cell functions.     

In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fiber mats

In the present contribution, the potential for use of the ultrafine electrospun fiber mats of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as scaffolding materials for skin and nerve regeneration was evaluated in vitro using mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) as reference cell lines. Comparison was made with PHB and PHBV films that were prepared by solution-casting technique. Indirect cytotoxicity assessment of the as-spun PHB and PHBV fiber mats with mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) indicated that the materials were acceptable to both types of cells. The attachment of L929 on all of the fibrous scaffolds was significantly better than that on both the film scaffolds and tissue-culture polystyrene plate (TCPS), while RT4-D6P2T appeared to attach on the flat surfaces of TCPS and the film scaffolds much better than on the rough surfaces of the fibrous scaffolds. For L929, all of the fibrous scaffolds were superior in supporting the cell proliferation to the film counterparts, but inferior to TCPS at days 3 and 5, while, for RT4-D6P2T, the rough surfaces of the fibrous scaffolds appeared to be very poor in supporting the cell proliferation when comparing with the smooth surfaces of TCPS and the film scaffolds. Scanning electron microscopy was also used to observe the behavior of both types of cells that were cultured on both the fibrous and the film scaffolds and glass substrate for 24 h.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

In vitro evaluation of an RGD-functionalized chitosan derivative for enhanced cell adhesion

Tissue repair is a spontaneous process that is initiated on wounding. However, if this complex mechanism is impaired or not sufficient the use of biomaterials might increase the chance of successful healing. In this view, an RGD-functionalized polymer was developed to promote dermal healing. A water-soluble chitosan derivative, carboxymethyl-trimethylchitosan (CM-TM-chitosan) was synthesized and GRGDS-moieties were grafted to the backbone at a concentration of 59nmol/mg polymer to increase cell-biomaterial interaction. Tested in vitro with cultured human dermal fibroblasts, the developed polymer showed good biocompatibility and the initial adhesion was increased by 3-5 times due to the GRGDS-moieties. Moreover, cell spreading was specific to the interaction with GRGDS, giving a 12-fold increase of cells showing a fully spread morphology within 30min. Overall, CM-TM-chitosan conjugated with GRGDS-peptides may prove useful as a biomaterial in wound healing.     

Cell type-specific binding of Ricinus lectin to murine cerebellar cell surfaces in vitro

Summary The binding of several plant lectins, Concanavalin A (ConA), Lens culinarisA (LCA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin 120 (RCA120) to cell surfaces of developing mouse cerebellar cells was assayed by the use of fluorescein isothiocyanate (FITC)-conjugated compounds. Freshly dissociated, live single-cell suspensions from 6-day-old mouse cerebellum contain 93% ConA, 99% LCA, 98% WGA, and 59% RCA 120-positive cells with ring fluorescence. Of the RCA 120-positive cells, 4% express a high and 55% a lower or very low number of lectin receptors. Flow cytometric analysis of fluorescent lectin binding yields results qualitatively similar to those obtained by scoring positive and negative cells in the fluorescence microscope.In monolayer cultures of 6-day-old mouse cerebellum practically all cells express receptors for ConA, LCA, and WGA, whereas RCA 120 binding sites are absent from neurons with small cell bodies (granule, basket and stellate cells) and present in large number on neurons with large cell bodies (Purkinje and possibly Golgi Type-II cells) and fibroblasts. RCA 120 receptors are weakly expressed on astro-and oligodendroglia. Cell type-specific expression of RCA 120 receptors is constant throughout all ages studied (embryonic day 13 to postnatal day 9). At early embryonic ages the proportion of highly fluorescent neurons with large cell bodies is significantly increased.     

Chitosan Treatment Delays the Induction of Senescence in Human Foreskin Fibroblast Strains

Fibroblasts have been extensively used as a model to study cellular senescence. The purpose of this study was to investigate whether the human foreskin fibroblast aging process could be regulated by using the biomaterial chitosan. Fibroblasts cultured on commercial tissue culture polystyrene (TCPS) entered senescence after 55–60 population doublings (PDs), and were accompanied by larger cell shape, higher senescence-associated β-galactosidase (SA β-gal) activity, lower proliferation capacity, and upregulation of senescence-associated molecular markers p21, p53, retinoblastoma (pRB), and p16. Before senescence was reached, PD48 cells were collected from TCPS and seeded on chitosan for three days (PD48-Cd3) to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in PD48-Cd3 spheroids were downregulated significantly. Following chitosan treatment, fibroblasts reseeded on TCPS showed lower SA β-gal activity, increased cellular motility, and a higher proliferation ability of 70–75 PDs. These phenotypic changes were not accompanied by colonies forming in soft agar and a continuous decrease in the senescence-associated proteins p53 and pRB which act as a barrier to tumorigenesis. These results demonstrate that chitosan treatment could delay the induction of senescence which may be useful and safe for future tissue engineering applications.     

Addition of biological functionality to poly(epsilon-caprolactone) films

Biodegradable polyesters such as poly(epsilon-caprolactone) (PCL) have a number of biomedical applications; however, their usage is often limited by a lack of biological functionality. In this paper, a PCL-based polymer containing pendent groups activated by 4-nitrophenyl chloroformate (NPC) and reactive toward primary amines has been cast into thin films. The reactivity of the films toward poly(l-lysine) and the cell adhesion peptide, GRGDS, was assessed, and their cell adhesive capabilities were characterized. ATR-FTIR analysis found that NPC functional groups were present on the surface of the cast film, and the synthesis, conjugation, and visualization of a fluorescent molecule on these films further demonstrated the success of this functionalization methodology. The immersion of these films into a solution of either poly(l-lysine) (PLL) or GRGDS in PBS (pH 7.4) and subsequent 3T3 fibroblast adhesion studies demonstrated significant improvement in cell adhesion and spreading over films cast from unmodified PCL. This investigation has shown that this novel NPC-containing polymer can be utilized in many applications where increased cellular adhesion is required, or the coupling of specific molecules to polymer surfaces is of interest.     

Lectin-mediated attachment and ingestion of yeast cells and erythrocytes by hamster fibroblasts

Yeast cell attachment to Concanavalin A (ConA)-coated fibroblasts depends on the degree of saturation of ConA-binding sites on the fibroblast. Under comparable conditions, fresh mouse erythrocytes fail to establish stable contacts with ConA-coated fibroblasts. The interaction of ConA-coated erythrocytes with fibroblasts and of non-coated erythrocytes with wheat germ agglutinin (WGA)-coated fibroblasts is remarkably less efficient than that of yeast cells interacting with ConA-coated fibroblasts. Ingestion of attached cells was not observed in any of the above lectin-mediated cell-cell interactions. Yeast cells coated with ConA show a high extent of attachment to fibroblasts (three-fold that of yeast cell attachment to ConA-coated fibroblasts). The attachment is highly temperature sensitive, being 3 times more at 37 °C than at 14 °C. A significant fraction of attached yeast cells (˜46%) is ingested by the fibroblasts during the 60 min incubation at 37 °C. The ingestion exhibits a strong temperature dependence, being nil at 14 °C and amounting to 150 and 600 ingested yeast cells per 100 fibroblasts at 24 °C and 37 °C, respectively. Transmission and scanning electron microscopy of ConA-mediated yeast cell-fibroblast interaction indicates a tighter interaction when the yeast cells are coated with ConA than when the fibroblasts are coated with ConA. Thus spreading of the plasma membrane around the attached yeast cell as well as transduction of attachment to ingestion could be triggered only under conditions of a very extensive multibridge interaction between the two apposing surfaces. Such an interaction is not achieved when the mobility of ConA-receptors within the fibroblast membrane plane is restricted as a result of crosslinking with ConA.     

Upregulation of HSP72 attenuates tendon adhesion by regulating fibroblast proliferation and collagen production via blockade of the STAT3 signaling pathway

The proliferation of fibroblasts creates an environment favoring post-operative tendon adhesion, but targeted therapy of this pathology remains in its infancy. In this study, we explored the effect of heat shock protein 72 (HSP72), a major inducible member of the heat shock protein family that can protect cells against many cellular stresses including heat shock, on fibroblast proliferation in tendon adhesion, with its underlying mechanisms investigated. HSP72 expression was examined in an established rat model of tendon injury using RT-qPCR and immunoblot analysis. After conducting ectopic expression and depletion experiments in fibroblast NIH3T3 cells, we determined the effects of HSP72 on the expression of α-SMA and STAT3 signaling pathway-related genes, fibroblast proliferation, as well as collagen production. The mRNA (65.46%) and protein (63.65%) expression of HSP72 was downregulated in the rat model of tendon injury. The in vitro experiments revealed that overexpression of HSP72 inhibited fibroblast proliferation (42.57%) and collagen production (45.60%), as well as reducing α-SMA expression (42.49%) and the extent of STAT3 phosphorylation (55.46%). Moreover, we observed that HSP72 overexpression reduced inflammation as well as the number of inflammatory cell infiltration and fibroblasts in vivo. Furthermore, the inhibited extent of STAT3 phosphorylation contributed to the impaired fibroblast proliferation and collagen production evoked by upregulated HSP72. In summary, the present study unveils an inhibitory role of HSP72 in tendon adhesion via inactivation of the STAT3 signaling pathway. This finding may enable the development of new therapeutic strategies for the prevention against tendon adhesion.     

Lectin-modified trifunctional nanobiosensors for mapping cell surface glycoconjugates

Nanomaterial-based nanobiosensors (nanobiodevices or nanobioprobes) are increasingly emphasized. Here, quantum dots and gamma-Fe(2)O(3) magnetic nanoparticles were co-embedded into single swelling poly(styrene/acrylamide) copolymer nanospheres to fabricate fluorescent-magnetic bifunctional nanospheres. Subsequently, fluorescent-magnetic-biotargeting trifunctional nanobiosensors (TFNS) modified with wheat germ agglutinin (WGA), peanut agglutinin (PNA) or Dolichos biflorus agglutinin (DBA) were conveniently produced so as to bind with A549 cells which are surface-expressed with N-acetylglucosamine, d-galactosamine and N-acetylgalactosamine residues. The values of WGA, PNA and DBA on each nanobiosensor were calculated to be 40, 14 and 60, respectively. These three kinds of lectin-modified trifunctional nanobiosensors (lectin-TFNS) can be used for qualitative and quantitative analysis of the glycoconjugates on A549 cell surface. The fluorescence intensity of WGA-modified nanobiosensors related to N-acetylglucosamine on A549 cell surface was much higher than that of PNA-modified nanobiosensors corresponding to d-galactosamine and that of N-acetylgalactosamine-related DBA-modified nanobiosensors, which is consistent with the results detected by flow cytometry. Lectin-modified trifunctional nanobiosensors not only can quantify the different glycoconjugates on A549 cell surface, but also can recognize and isolate A549 cells. 0.5mg of WGA-modified fluorescent-magnetic trifunctional nanobiosensors could capture 7.0 x 10(4) A549 cells. Therefore, the lectin-modified trifunctional nanobiosensors may be applied in mapping the glycoconjugates on cell surfaces and for recognition and isolation of targeted cells.     

Interactions of lectins and monoclonal antibodies with human mononuclear cells. I. Specific inhibition of OKT4 and OKT8 binding by Ricinus communis agglutinin and wheat germ agglutinin

We used flow cytometry to examine effects of lectins on interactions between human lymphocytes and the anti-T cell monoclonal reagents OKT4 (T helper-specific) and OKT8 (T suppressor-specific). Wheat germ agglutinin (WGA) inhibited OKT8 binding to lymphocytes by a mean 77% and Ricinus communis agglutinin (RCA-I) inhibited OKT4 binding by 66%. Inhibition was abolished in each case by appropriate carbohydrate hapten inhibitors of lectin binding, indicating it was mediated by the lectin saccharide combining sites. Neither WGA nor RCA-I inhibited binding of OKT3, a pan-T cell monoclonal reagent. In addition, a group of other lectins with a variety of nominal carbohydrate specificities did not inhibit OKT4 or OKT8 binding. Preincubation experiments and gel filtration indicated that inhibition in each case was due to competition between lectin and monoclonal for binding to cell surfaces, not to direct lectin-monoclonal antibody interactions. Treatment of lymphoid cells with OKT8 and complement reduced OKT8- and WGA-binding cells concurrently, whereas treatment with OKT4 and complement did not reduce percentages of either type of cell. Similarly, specific depletion of OKT8-binding cells abolished the mitogenic response to WGA but not that to PHA. Cell populations enriched for WGA-binding cells prepared by flow cytometry and cell sorting demonstrated parallel enrichment for OKT8-binding and depletion of OKT4-binding cells. Therefore, these data demonstrate specific inhibition of OKT4 and OKT8 binding by the lectins, RCA-I and WGA, respectively. Inhibition was mediated by lectin binding to lymphoid cell surfaces, perhaps directly to the T4 or T8 antigens. The observations indicate that lectins may prove useful for investigating structural features of some immunologic cell surface markers. Furthermore, they provide the possibility that certain in vitro effects of lectins on immune function may result from their interactions with molecules such as the T4 and T8 antigens.     

Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films

Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.     

A novel apoptotic pathway as defined by lectin cellular initiation

In this study of lectin-induced apoptosis we found that wheat germ agglutinin (WGA) initiated an accelerated type of programmed cell death developing after only 30 min of incubation with tumor cells. To analyze possible mechanisms, studies were focused using the WGA lectin whose carbohydrate specificity is well defined. We found that WGA could induce apoptosis by binding to either N-acetylneuraminic acid or N-acetylglucosamine (GlcNAc) on the cell surface of normal and malignant cells. We also showed that it is unlikely that WGA triggers apoptosis by binding to the carbohydrate portion of Fas. CrmA gene transfection did not inhibit WGA-mediated apoptosis of Jurkat cells. In addition, Jurkat-R cells selected for resistance to Fas signaled apoptosis manifested high sensitivity to WGA as did Fas-negative BL6 melanoma cells. WGA-induced apoptosis is also caspase-3-independent and was found to be triggered via a mitochondrial pathway. WGA induced a loss of transmembrane potential, disruption of the inner mitochondria membrane, and release of cytochrome c and caspase-9 activation after 30 min of cell interaction. Interestingly, Bcl-2 gene transfection did not affect sensitivity of Jurkat cells to WGA. The Jurkat-R subline that has been shown to be Bax and Bak deficient and resistant to various apoptotic signals was highly sensitive to WGA-induced apoptosis. In summary, WGA triggers a unique pattern of apoptosis that is extremely fast, Fas- and caspase-3-independent, and is mediated via a mitochondrial pathway. However, its mitochondrial component is unrestrained by the loss of Bax and Bak or the upregulation of Bcl-2 expression.     

The Inhibitory Effect of an RGD-Human Chitin-Binding Domain Fusion Protein on the Adhesion of Fibroblasts to Reacetylated Chitosan Films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation, and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). A method based on the use of a carbohydrate-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyze the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitin-binding domain.     

Improving biomaterial properties of collagen films by chemical modification

Films of bovine collagen were chemically modified with the goal of improving their biomaterial properties. The modified films were investigated with respect to their affinity to fibroblast and endothelial cells, as well as their antibacterial properties tested by adhesion of Staphylococcus aureus. Modifications that only change the net charge of collagen, such as acetylation, succinylation, and treatment with glutaraldehyde (all increase the negative charge), and amination with ethylenediamine (EDA), N,N-dimethyl-EDA (DMEDA), or butylamine (all increase the positive charge), did not dramatically alter the mammalian cell attachment to the film. In contrast, derivatization of collagen using methoxypoly(ethylene glycol) (PEG) diminished the attachment of fibroblasts by 98 +/- 1% and of endothelial cells by more than 99% compared to unmodified collagen. Moreover, the rate of growth of fibroblasts dropped by 97 +/- 1% and that of endothelial cells by 88 +/- 3% as a result of PEGylation of collagen. Adhesion of S. aureus cells also plummeted by 93 +/- 2% as a result of this PEGylation. With these antifouling properties, PEG-collagen may be a promising coating material for coronary stents. Subsequent derivatization of PEG-collagen with EDA or DMEDA abolished its mammalian cell-repelling ability, whereas bacterial cell repulsion was partially retained: for example, DMEDA-modified PEG-collagen exhibits up to a 5-fold lower bacterial adhesion than collagen. It is worth noting that a material that allows mammalian cell attachment but reduces bacterial adhesion could be useful as an implant or coating.     

Protein 1a: A major wheat germ agglutinin binding protein on the surface of human granulocytes associated with the cytoskeleton

Lectin-receptors on leukocyte and endothelial surfaces are becoming more important in the light of increasing evidence which implicates lectin-carbohydrate interactions in diverse physiological phenomena. This study reports the identification of a major 118 kDa granulocyte surface protein, (Protein 1a) which binds the lectin wheat germ agglutinin (WGA), and is distinctly different from reported WGA binding granulocyte membrane proteins. Protein 1a has been isolated from the Triton-soluble and Triton-insoluble lysates of normal individuals and patients with Chronic Myeloid Leukemia (CML) using a combination of differential solubilization, lectin affinity, ion exchange chromatography and HPLC. The protein from the detergent lysates of both normal and CML granulocytes has similar pI values, lectin affinities, and hydrophobicity. However, its solubility in Triton is different in the two cell types. In 71% of CML cases examined, Protein 1a exhibits decreased Triton solubility suggesting its increased association with the cytoskeleton (CSK). Stimulation of normal granulocytes with WGA leads to the translocation of the soluble form of Protein 1a to the Triton-insoluble fraction. This cytoskeletal recruitment of Protein 1a is sustained only under conditions of excess WGA and occupied receptor. The CSK disruptive agent dihydrocytochalasin B (H₂CB) releases the insoluble form of the receptor into the Triton-soluble fraction. Investigation of a CSK-involving process such as ligand internalization revealed that CML granulocytes exhibit slower kinetics of internalization of fluorescent WGA molecules. Since Protein 1a is a major WGA receptor on the granulocyte surface, its decreased Triton solubility in CML granulocytes suggests that this may be one of the factors contributing to the defective receptor-mediated endocytosis of WGA by CML cells, arising as a consequence of altered membrane-CSK interaction — a nodal point in the signal transduction cascade.     

N-acetylglucosamine and adenosine derivatized surfaces for cell culture: 3T3 fibroblast and chicken hepatocyte response

3T3 fibroblasts and primary chicken hepatocytes were cultured on derivatized polystyrene surfaces to examine the effect of cell-specific ligands on cellular morphology and growth. Surfaces were prepared by derivatizing chloromethylated polystyrene with N-acetylglucosamine (GlcNAc; recognized by the chicken asialoglycoprotein receptor) and adenosine (not recognized by adult hepatocytes). These surfaces were compared with tissue culture polystyrene (TCPS), acid-cleaned glass, and the unmodified chloromethylated polystyrene. The spreading, cytoskeletal structure and growth of the fibroblasts following attachment to these surfaces were examined. The extent of attachment, total protein levels, and DNA contents for surfaces-attached chicken hepatocytes were also measured. Fibroblast spreading was greatest on polymer surfaces derivatized with GlcNAc, whereas cytoskeletal structure and growth rate were independent of surface chemistry. Although chicken hepatocytes attached most efficiently to the GlcNAc derivatized polymer, the total protein and DNA levels of the surface-attached cells were not affected. In anticipation of the application of these polymers for cell culture and hybrid artificial organ design, the GlcNAc-derivatized polystryrene was fabricated into porous microcarriers. Fibroblasts grew avidly on the microcarriers, whereas chicken hepactocytes adhered well to the formed large aggregates arounds the microcarriers.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae. A light- and electron-microscopic study

The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone which was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.