Does glycosylation of melanoma cells influence their interactions with fibronectin?

Cell surface integrins, especially those binding to fibronectin (FN), participate in processes of tumor cell invasion and metastasis. Changes in glycosylation of cell surface adhesion proteins are often associated with malignant transformation of cells. In this study we examined the influence of swainsonine (SW) on adhesion, wound healing and haptotactic migration on FN, comparing the responses of different human melanoma cell lines: primary WM35 and metastatic WM9, WM239 and A375. We also examined the role of alpha subunits in adhesion to FN. All of the antibodies inhibited adhesion to FN but with different efficiencies depending on the cell line. Adhesion was mediated mainly by integrin alpha(5)beta(1) (WM9, A375), alpha(3)beta(1) (WM35, A375, WM239). Scratch wound repair was significantly faster on FN-coated wells than on plastic for all cells except for WM9. A375 and WM9 had the greatest migration ability, both expressing the highest level of alpha(5)beta(1) integrin. It seems very likely that adhesion to FN can be accomplished by many different integrins, but for effective migration alpha(5)beta(1) integrin is responsible. Only A375 and WM239 cell lines reacted to SW treatment. In the presence of SW WM239 and A375 cells had 70% and 40% increased adhesion to FN, and their migration was decreased 40% and 50%, respectively. Interestingly, although most of the cell lines share a common profile of integrins, each line interacted with FN differently. They differed mainly in the repertoire of integrins used for adhesion, and in the manner in which glycosylation affected these processes. The influence of SW was observed in two metastatic cell lines indicating the contribution of glycosylation status to the progression of melanoma. The lack of reaction to SW in WM9 cells may suggest that there is a threshold in the expression level of the highly branched N-glycans that may influence the adhesion and migration properties of the cell.     

A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.     

Structure of full-length bacterial chitinase containing two fibronectin type III domains revealed by small angle X-ray scattering

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 consists of an N-terminal catalytic domain, two fibronectin type III domains (FnIIIDs), and a C-terminal chitin-binding domain. The full-length structure of ChiA1 was studied by small angle X-ray scattering. The obtained low-resolution structure showed that ChiA1 is an elongated molecule with a length of approximately 145 A composed of a large globular head and a rod-like tail. Combination with known high-resolution structures of individual ChiA1 domains provided a model of the domain arrangement. In this model, two FnIIIDs connect to each other in an extended rod-like shape without large bending between the FnIIIDs, and contribute largely to the length of ChiA1.     

Oligomeric forms of peptide fragment PrP(214-226) in solution are preferentially recognized by PrP(Sc)-specific antibody

A specific monoclonal antibody (mAb) V5B2 that discriminates between brain tissue of Creutzfeldt-Jakob disease patients and that from normal controls without proteinase K digestion has been prepared using a 13-residue synthetic peptide P1 from the primary structure of human PrP. In the light of the specific interaction between mAb V5B2 and the pathological isoform of PrP (PrP(Sc)), we investigated the solution behavior of antigen P1 and its interactions with mAb V5B2. Our results show that V5B2 recognizes epitope P1 in dimeric/oligomeric forms in solution and in the fibril-like aggregates, as well as in PrP(Sc) aggregates, and demonstrate that the specific epitope is present in all of these forms, but not in PrP(C).     

Aggregating the amyloid Abeta(11-25) peptide into a four-stranded beta-sheet structure

We present a detailed analysis of the structural properties of one monomer of Abeta(11-25) as well as of the aggregation mechanisms for four chains of Abeta(11-25) using the activation-relaxation technique coupled with a generic energy potential. Starting from a random distribution of these four chains, we find that the system assembles rapidly into a random globular state that evolves into three- and four-stranded antiparallel beta-sheets. The aggregation process is considerably accelerated by the presence of preformed dimers. We also find that the reptation mechanism already identified in shorter peptides plays a significant role here in allowing the structure to reorganize without having to fully dissociate.     

Insights into the architecture of the Ure2p yeast protein assemblies from helical twisted fibrils

The protein Ure2 from baker's yeast is associated with a heritable and transmissible phenotypic change in the yeast Saccharomyces cerevisiae. Such prion properties are thought to arise from the fact that Ure2p is able to self-assemble into insoluble fibrils. Assemblies of Ure2p are composed of full-length proteins in which the structure of the globular, functional, C-terminal domain is retained. We have carried out structural studies on full-length, wild-type Ure2p fibrils with a regularly twisted morphology. Using electron microscopy and cryo-electron microscopy with image analysis we show high-resolution images of the twisted filaments revealing details within the fibrillar structure. We examine these details in light of recent proposed models and discuss how this new information contributes to an understanding of the architecture of Ure2p yeast prion fibrils.     

Fibronectin and Focal Adhesion Kinase Small Interfering RNA Modulate Rat Retinal Müller Cells Adhesion and Migration

Retinal Müller cells (RMCs) hypertrophy and proliferation play a crucial role in epiretinal membrane formation. This study was designed to analyze the effects of Fibronectin and specific FAK siRNA in cell adhesion and migration in rat Müller cells. RMCs were cultured and identified by GFAP, Vimentin, and GLAST mAb, respectively. The cells were planted on dishes coated with Fibronectin at 0, 1, 5, 10, 50, and 100 μg/ml. The attachment and migration assay was applied to characterize the RMCs–Fibronectin interactions. Cell lysis and Western blotting were utilized to detect β₁-integrin, FAK, and GLAST protein expression. Then the cells were treated with FAK siRNA, non-targeting siRNA, and control medium. The cell cycle and apoptosis rate was determined by flow cytometry. The attachment, migration, and Western blotting assay were repeated. These data suggested that almost all the cells expressed GFAP, Vimentin, and GLAST, respectively, which ensured most of the harvested cells were RMCs. In attachment assay, the A570 values increased significantly with time (F = 1105.439, P < 0.001) and Fibronectin concentration (F = 424.683, P < 0.001). There were significant difference between each Fibronectin concentration in RMCs migration (F = 34.703, P < 0.000). The expression ratio of FAK, β₁-integrin, and GLAST elevated significantly as Fibronectin concentration increased (F = 54.755, P < 0.000; F = 119.962, P < 0.000; F = 39.287, P < 0.000). The Fibronectin pretreatment was settled on 50 μg/ml for siRNA inhibition assays. The specific FAK siRNA treatment significantly increased G₀/G₁ percentage and apoptosis rate compared with NT siRNA and control group (F = 11.526, P = 0.009; F = 64.772, P < 0.000). The apoptotic rate was significantly suppressed by inhibitors of caspase-8 and 3 (F = 10.500, P = 0.011). The A570 values were significantly suppressed in FAK siRNA groups compared with NT siRNA and control group (F = 154.241, P < 0.000), and the mean migratory cells per view field were significantly decreased (F = 10.906, P = 0.001). FAK and GLAST expression ratio decreased significantly after FAK siRNA treatment (F = 5.315, P = 0.047; F = 5.985, P = 0.042). Take together, FAK is involved in β₁-integrin mediated adhesive signaling and play a critical role in regulating Müller cell adhesion, migration, and so far as to glutamate transportation functions.     

Myocilin promotes substrate adhesion,spreading and formation of focal contacts in podocytes and mesangial cells

Myocilin, a secreted glycoprotein of the olfactomedin family, is constitutively expressed in podocytes of the rat kidney and induced in mesangial cells during mesangioproliferative glomerulonephritis. As myocilin has been found to be associated with fibrillar components of the extracellular matrix, and adhesive properties have been shown for other members of the olfactomedin family, we hypothesized that myocilin might play a role in cell-matrix interactions in the glomerulus. To elucidate functional properties of myocilin, recombinant myocilin was expressed in 293 EBNA cells and purified by Ni-chelate and heparin chromatography. Culture plates were coated with myocilin, and primary rat mesangial cells and cells from an immortal murine podocyte cell line were seeded onto the plates in serum free conditions. Both cell types showed concentration-dependant attachment to myocilin, an effect that was statistically significant and could be blocked with specific antibodies. When compared to equal amounts of fibronectin or collagen 1, myocilin was less effective in promoting substrate adhesion. Synergistic effects in substrate adhesion were observed when myocilin was added to low concentrations of fibronectin. Twenty-five percent of cells that had attached to myocilin substrates showed spreading and expressed focal contacts which were labeled by vinculin/phalloidin staining. Comparable findings were observed when human or murine trabecular meshwork cells were seeded on myocilin substrates. Adhesive properties of myocilin required multimer formation, and were not observed when culture plates were coated with a C-terminal fragment of myocilin, containing the olfactomedin domain. We conclude that myocilin promotes substrate adhesion of podocytes and mesangial cells, and might contribute to cell-matrix adhesion of both cell types in vivo.     

The Critical Aggregation Concentration of β-Lactoglobulin-Based Fibril Formation

The critical aggregation concentration (CAC) for fibril formation of β-lactoglobulin (β-lg) at pH 2 was determined at 343, 353, 358, 363, and 383 K using a Thioflavin T assay and was approximately 0.16 wt%. The accuracy of the CAC was increased by measuring the conversion into fibrils at different stirring speeds. The corresponding binding energy per mol, as determined from the CAC, was 13 RT (∼40 kJ mol⁻¹) for the measured temperature range. The fact that the CAC was independent of temperature within the experimental error indicates that the fibril formation of β-lg at pH 2 and the measured temperature range is an entropy-driven process.