Neil A. Richardson

Characterisation of cellulase activity in the digestive system of the redclaw crayfish (Cherax quadricarinatus)

Abstract Endogenous cellulase activity was identified in the gastric fluid and digestive gland of the redclaw crayfish. Cellulase showed maximal activity from pH 4 to 5 and was stable for up to 2 h at 40°C. Cellulase activity in the digestive gland was unaffected by antibiotic treatment. Taken together these findings suggest a significant endogenous component for redclaw cellulase activity. Partial purification of cellulase activity was performed using anion exchange and gel filtration chromatography. One major and one minor band of activity were identified subsequently by SDS-PAGE and zymography. The molecular weight of the major band was estimated at 40 kDa while the minor band was estimated at 30 kDa. Redclaw cellulase enzymes demonstrated broad substrate specificity, hydrolysing polysaccharides containing β-1,4 and mixed β-1,4 and β-1,3 glycosidic bonds but showed a preference for soluble substrates. Hydrolysis products of cellodextrins of various lengths also showed that the enzymes liberated free glucose. Exposure of redclaw to antibiotics resulted in a dramatic decline in bacterial populations in the gastric contents (>90%) but only a 40% decline in cellulase activity.

ORGANIZATION, SEQUENCE, AND EXPRESSION OF THE GENE ENCODING IGFII FROM BARRAMUNDI (TELEOSTEII; LATES CALCARIFER)

We have characterized the gene encoding IGFII from the teleost fish species barramundi, Lates calcarifer. The barramundi gene spans 5.5 kb of DNA and comprises four exons and three introns. The barramundi and salmon IGFII genes share >85% sequence similarity across all the exons and also share some regions of high sequence identity within the promoter regions and the introns. The mature barramundi IGFII peptide comprises 70 amino acid residues and shares 84 and 96% similarity with salmonid and seabream IGFII, respectively, with the majority of replacements located in regions cleaved from the mature peptide. IGFII mRNA transcripts were detected in liver, muscle, intestine, gill, heart, and brain from juvenile barramundi. This distribution mirrors that seen in the rainbow trout and seabream and extends the tissue types which synthesize IGFII in fish to include the intestine.

LOCALISATION OF INSULIN-LIKE GROWTH FACTOR-I (IGF-I) IMMUNOREACTIVITY IN THE OLIVOCEREBELLAR SYSTEM OF DEVELOPING AND ADULT RATS

The molecular mechanisms which underlie the development of the olivocerebellar topography are not fully understood. Insulin-like growth factor-I (IGF-I) is a growth factor known to play important roles in neural development and it has been identified within the cerebellum and the inferior olive. To assess the contribution of IGF-I to the development of climbing fibre topography, the distribution of IGF-I-like immunoreactivity (IGF-I IR) was identified in the cerebellar cortex and inferior olive of rats, 0, 3, 5, 7, 10, 15, 21, 28 and 90 days old. In the cerebellar cortex, IGF-I IR was localised solely to Purkinje cells and its distribution was spatially and temporally regulated in a manner which coincides with climbing fibre development. At birth, weak IGF-I IR was detected in a few Purkinje cells in the ventral vermis. More Purkinje cells became positive until at postnatal day 7(P7) all Purkinje cells displayed IGF-I IR. Subsequently, a subpopulation of Purkinje cells lost their reactivity for IGF-I to leave IGF-I-positive cells organised into sagittal bands by P15. IGF-I IR was also seen in all subdivisions of the inferior olive between birth and P10 in a distribution which paralleled the maturation of the inferior olive. The Purkinje cell and inferior olivary IGF-I IR parallels climbing fibre development and thus the results of this study support the hypothesis that IGF-I is involved in the development of climbing fibre topography.

Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial (HCE) cells. Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell-attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favour the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

In vitro characterization and in vivo clearance of recombinant barramundi (Lates calcarifer) IGF-I

Little is known about fish insulin-like growth factors (IGFs) as only small amounts have been isolated from native sources or indeed produced recombinantly. This report describes the production of milligram quantities of recombinant barramundi IGF-I (bIGF-I) and its subsequent characterization. Recombinant bIGF-I was produced in Escherichia coli using a gene fusion system similar to that previously described for the production of other non-mammalian IGFs. Recombinant bIGF-I was similar to human IGF-I (hIGF-I) in stimulating protein synthesis and in competing for binding of labelled hIGF-I to IGF receptors whether tested in rat myoblasts or in salmon embryo fibroblasts. However, recombinant bIGF-I differed from its human counterpart in its affinity for a polyclonal antibody raised against hIGF-I, with at least 200-fold more bIGF-I required to obtain 50% displacement of labelled hIGF-I from the antibody. Hence, the recombinant protein will be essential for developing a specific homologous immunoassay for measuring IGF-I concentrations in barramundi during growth and development. In addition, studies investigating the clearance of labelled bIGF-I and hIGF-I in vivo reveal that the human protein is cleared from the circulation of juvenile barramundi almost twice as fast as the barramundi protein, thus providing the first in vivo evidence that there are functional differences between fish and human IGF-Is. Neutral gel chromatography of serum from the clearance study suggest that this is due to differences in the affinities of the labelled human and fish IGF-I for the IGFBPs present in barramundi.

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood–retinal barrier of patients afflicted with age‐related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruchs membrane (3 µm). Confluent cultures of RPE cells (ARPE‐19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell®). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F‐actin, ZO‐1, ezrin, cytokeratin pair 8/18, RPE‐65 and Na+/K+‐ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright

Evaluation of Eph receptor and ephrin expression within the human cornea and limbus

Eph receptor tyrosine kinases and their ligands, the ephrins, regulate the development and maintenance of multiple organs but little is known about their potential role within the cornea. The purpose of this study was to perform a thorough investigation of Eph/ephrin expression within the human cornea including the limbal stem cell niche. Initially, immunohistochemistry was performed on human donor eyes to determine the spatial distribution of Eph receptors and ephrins in the cornea and limbus. Patterns of Eph/ephrin gene expression in (1) immortalised human corneal endothelial (B4G12) or corneal epithelial (HCE-T) cell lines, and (2) primary cultures of epithelial or stromal cells established from the corneal limbus of cadaveric eye tissue were then assessed by reverse transcription (RT) PCR. Limbal epithelial or stromal cells from primary cultures were also assessed for evidence of Eph/ephrin-reactivity by immunofluorescence. Immunoreactivity for ephrinA1 and EphB4 was detected in the corneal endothelium of donor eyes. EphB4 was also consistently detected in the limbal and corneal epithelium and in cells located in the stroma of the peripheral cornea. Expression of multiple Eph/ephrin genes was detected in immortalised corneal epithelial and endothelial cell lines. Evidence of Eph/ephrin gene expression was also demonstrated in primary cultures of human limbal stromal (EphB4, B6; ephrinA5) and epithelial cells (EphA1, A2; ephrinA5, B2) using both RT-PCR and immunofluorescence. The expression of Eph receptors and ephrins within the human cornea and limbus is much wider than previously appreciated and suggests multiple potential roles for these molecules in the maintenance of normal corneal architecture.

Incorporation of Human Recombinant Tropoelastin into Silk Fibroin Membranes with the View to Repairing Bruch’s Membrane

Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

Demonstration of P-selectin expression and potential function in human corneal epithelial cells

&NA; In response to an unexpected observation of apparent localisation by immunocytochemistry, we have investigated the potential expression and function of P‐selectin (CD62P) in human corneal epithelial cells. The SV40 immortalised cell line, HCE‐T (validated by STR profiling), along with multiple donor corneal‐limbal tissue samples, were examined for P‐selectin expression using a combination of immunocytochemistry, Western blotting, RT‐PCR and immunohistochemistry. Potential expression of the major ligand for P‐selectin (P‐selectin glycoprotein ligand‐1; PSGL‐1; CD162) was also examined by immunocytochemistry and RT‐PCR. A selective inhibitor of P‐selectin‐PSGL‐1 binding (KF38789) was subsequently tested for effects on HCE‐T cells using a cell culture gap‐closure assay. HCE‐T cells as well as primary epithelial cultures derived from donor corneal‐limbal tissue, displayed positive immunostaining for P‐selectin. Staining was particularly evident at cell‐cell boundaries and at the outer edge of expanding epithelial islands. P‐selectin expression was confirmed by Western blotting and RT‐PCR (validated by product sequencing), as well as by immunohistochemistry performed on serial sections of corneal‐limbal tissue stained for P‐selectin, keratin 3 and p63. PSGL‐1 was detected by RT‐PCR and immunocytochemistry in both corneal epithelial cells as well as human limbal fibroblasts (HLF). KF38789 (5 &mgr;M) significantly reduced closure of a 500‐&mgr;m gap between confluent sheets of HCE‐T cells over an 8‐hr period (by ˜40%, p < 0.01; paired two‐tailed T test), but had no effect on culture gap‐closure by either HLF or murine 3T3 fibroblasts. These results provide evidence of P‐selectin expression in human corneal epithelial cells and suggest a potential role for this glycoprotein in facilitating the net movement of confluent sheets of human corneal epithelial cells. HighlightsHuman corneal epithelial (HCE) cells express P‐selectin in vitro and in situ.HCE and limbal fibroblasts both appear to express the P‐selectin ligand, PSGL‐1.An inhibitor of P‐selectin binding to PSGL‐1 reduces HCE migration in vitro.These findings suggest a potential new role for P‐selectin within the cornea.

Optimization of Corneal Epithelial Progenitor Cell Growth on Bombyx mori Silk Fibroin Membranes

Scaffolds prepared from silk fibroin derived from cocoons of the domesticated silkworm moth Bombyx mori have demonstrated potential to support the attachment and growth of human limbal epithelial (HLE) cells in vitro. In this study, we attempted to further optimize protocols to promote the expansion of HLE cells on B. mori silk fibroin- (BMSF-) based scaffolds. BMSF films were initially coated with different extracellular matrix proteins and then analysed for their impact on corneal epithelial cell adhesion, cell morphology, and culture confluency. Results showed that collagen I, collagen III, and collagen IV consistently improved HCE-T cell adherence, promoted an elongated cell morphology, and increased culture confluency. By contrast, ECM coating had no significant effect on the performance of primary HLE cells cultured on BMSF films. In the second part of this study, primary HLE cells were grown on BMSF films in the presence of medium (SHEM) supplemented with keratinocyte growth factor (KGF) and the Rho kinase inhibitor, Y-27632. The results demonstrated that SHEM medium supplemented with KGF and Y-27632 dramatically increased expression of corneal differentiation markers, keratin 3 and keratin 12, whereas expression of the progenitor marker, p63, did not appear to be significantly influenced by the choice of culture medium.