Perfusion culture enhanced human endometrial stromal cell growth in alginate-multivalent integrin α5β1 ligand scaffolds.

A method to functionalize alginate by introducing monomeric or self-assembling (tetrameric) fibronectin (FN) domains is described, leading to a functional scaffold, which is used for three dimensional (3D) culture of human endometrial stromal cells (EnSCs). EnSCs encapsulated in the functional alginate were cultured under perfusion using the TissueFlex® platform, a multiple parallel microbioreactor system for 3D cell culture. The effect of the novel scaffold and the effect of perfusion were examined.

Clustered integrin α5β1 ligand displays model fibronectin-mediated adhesion of human endometrial stromal cells.

Progress towards endometrial tissue engineering for modelling endometrial diseases and infertility is frustrated by the inability to mimic the fibronectin (FN) extracellular matrix required by human endometrial stromal cells (EnSCs). Here we show that this is because of the requirement to present integrin α5β1 (the FN receptor) ligands in specifically oriented, polyvalent displays; by engineering controlled self-assembly of the 9th-10th type III FN domain pair (FIII9-10, the minimal integrin α5β1 ligand) immobilised in a specific orientation to cell culture surfaces. The fraction of adherent EnSCs seen to spread increased significantly for the multimeric ligand surfaces in the order: tetramer>trimer>dimer>monomer.

Silica condensation by a silicatein α homologue involves surface-induced transition to a stable structural intermediate forming a saturated monolayer.

Silicatein α exists within the protein filament of silica spicules of the marine sponge Tethya aurantium in a predominantly β-sheet structure. However, it is produced in a soluble form with mixed α-helix/β-sheet structure akin to its cathepsin L homologue. To understand this conformational transition in the context of enzyme catalyzed silica condensation, we used a functional, recombinant silicatein α termed 4SER.

Dimeric integrin alpha5beta1 ligands confer morphological and differentiation responses to murine embryonic stem cells.

We present the first report utilizing, and showing the functional relevance of, self-assembling polyvalent ligands specific for integrin alpha5beta1 in murine embryonic stem (mES) cell adhesion. Di, tri and tetrameric 9th-10th type III fibronectin domains (FIII9'10) were used to generate clustered integrin alpha5beta1 ligand surfaces for mES cell culture. Compared to gelatin, FIII9'10 (monomer), FIII9'10-trimer and -tetramer, the FIII9'10-dimer supported the highest number of mES cell colonies.

Oligomerisation and thermal stability of polyvalent integrin alpha5beta1 ligands.

Synthetic oligomeric integrin alpha5beta1 ligands, specifically immobilised to surfaces, facilitate increased fibroblast cell spreading compared with that associated with the monomer. These ligands consist of a N-terminal fibronectin domain pair, a spacer and a di-, tri- or tetrameric coiled coil. However, it is not yet clear what effect fusion of the fibronectin domains has on the predicted oligomerisation of the coiled coils.

IGFBP-3 and IGFBP-5 associate with the cell binding domain (CBD) of fibronectin.

We have used Surface Plasmon Resonance (SPR) - based biosensor technology to investigate the interaction of the six high affinity insulin-like growth factor binding proteins (IGFBP 1-6) with the cell binding domain (CBD) of fibronectin. Using a biotinylated derivative of the ninth and tenth TypeIII domains of FN ((9-10)FNIII), we show that IGFBP-3 and -5 bind to FN-CBD. We show that this binding is inhibited by IGF-I and that, for IGFBP-5, binding occurs through the C-terminal heparin binding domain of the protein.

A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations.

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions.

Protein-coated microcrystals for use in organic solvents: application to oxidoreductases.

Protein-coated microcrystals (PCMC), a biocatalyst preparation previously demonstrated to display substantially increased transesterification activity of proteases and lipases in organic solvents when compared to their as received counterparts [Kreiner M, Moore BD, Parker MC (2001) Chem. Commun. 12:1096--1097], was applied to oxidoreductases.

DNA-coated microcrystals.

Coprecipitation leads to self-assembly of bioactive DNA on the surface of salt, sugar or amino-acid crystals and provides a rapid inexpensive immobilization method suitable for preparing dry-powder formulations of nucleic acids, useful for storage, imaging and drug delivery.

High-activity biocatalysts in organic media: solid-state buffers as the immobilisation matrix for protein-coated microcrystals.

Recently, we reported a new high-activity biocatalyst for use in organic media termed protein-coated microcrystals (PCMC) (Kreiner et al. [2001] Chem Commun 12:1096-1097). These novel particles consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s) and are prepared in a one-step rapid dehydration process.

Morphological and enzymatic responses of a recombinant Aspergillus niger to oxidative stressors in chemostat cultures.

Continuous chemostat cultures of a recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme, were investigated with regard to their susceptibility to oxidative stress. The culture response to oxidative stress, produced either by addition of exogenous hydrogen peroxide (H(2)O(2)) or by high dissolved oxygen tension (DOT), was characterised in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). Since the morphology is so critical in submerged fungal bioprocesses, the key morphological indices were analysed using a semi-automated image analysis system.