Tony Vuocolo

Synthesis and properties of crosslinked recombinant pro-resilin

Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage; it is best known for its roles in insect flight and the remarkable jumping ability of fleas and spittle bugs. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast samples of this biomaterial may enable its use in situ for both industrial and biomedical applications.

Characterization of a Major Peritrophic Membrane Protein, Peritrophin-44, from the Larvae of Lucilia cuprina cDNA AND DEDUCED AMINO ACID SEQUENCES

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

Larvicidal activity of lectins on Lucilia cuprina: mechanism of action

Larvae of the blowfly Lucilia cuprina (Wied.) (Diptera: Calliphoridae) were grown in vitro on a serum‐free medium in the presence of a number of lectins. Lectins with specificities for β‐(1,4)‐N‐acetylglucosamine (wheat germ lectin) and α‐D‐mannopyranosyl and (α‐D‐glucopyranosyl residues (lentil lectin and Con A) caused strong concentration‐dependent inhibition of the growth of the larvae and substantial mortality. Wheat germ lectin had the strongest effects, showing 50% inhibition of larval growth at a concentration of 2 μM and 100% mortality at 25 μM. Other lectins with different sugar specificities had much less effect. The mechanism of the larvicidal action(s) of wheat germ lectin, lentil lectin and Con A was investigated. There were at least three effects of these lectins on L. cuprina larvae. First, these lectins bound to and reduced the permeability of the peritrophic membrane of the larvae. Second, they reduced ingestion of diet medium by larvae. Third, the lectins also bound to the apical membranes of larval gut epithelial cells although there were no obvious signs of damage to these cells. It is concluded that the combination of these effects probably results in the starvation of the larvae. The implications of these results in terms of possible control strategies for L. cuprina are discussed.

A highly elastic tissue sealant based on photopolymerised gelatin

Gelatin is widely used as a medical biomaterial because it is readily available, cheap, biodegradable and demonstrates favourable biocompatibility. Many applications require stabilisation of the biomaterial by chemical crosslinking, and this often involves derivatisation of the protein or treatment with cytotoxic crosslinking agents. We have previously shown that a facile photochemical method, using blue light, a ruthenium catalyst and a persulphate oxidant, produces covalent di-tyrosine crosslinks in resilin and fibrinogen to form stable hydrogel biomaterials. Here we show that various gelatins can also be rapidly crosslinked to form highly elastic (extension to break >650%) and adhesive (stress at break >100 kPa) biomaterials. Although the method does not require derivatisation of the protein, we show that when the phenolic (tyrosine-like) content of gelatin is increased, the crosslinked material becomes resistant to swelling, yet retains considerable elasticity and high adhesive strength. The reagents are not cytotoxic at the concentration used in the photopolymerisation reaction. When tested in vivo in sheep lung, the photopolymerised gelatin effectively sealed a wound in lung tissue from blood and air leakage, was not cytotoxic and did not produce an inflammatory response. The elastic properties, thermal stability, speed of curing and high tissue adhesive strength of this photopolymerised gelatin, offer considerable improvement over current surgical tissue sealants.

Identification of immune genes and proteins involved in the response of bovine mammary tissue to Staphylococcus aureus infection

BackgroundMastitis in dairy cattle results from infection of mammary tissue by a range of micro-organisms but principally coliform bacteria and Gram positive bacteria such as Staphylococcus aureus. The former species are often acquired by environmental contamination while S. aureus is particularly problematic due to its resistance to antibiotic treatments and ability to reside within mammary tissue in a chronic, subclinical state. The transcriptional responses within bovine mammary epithelial tissue subjected to intramammary challenge with S. aureus are poorly characterised, particularly at the earliest stages of infection. Moreover, the effect of infection on the presence of bioactive innate immune proteins in milk is also unclear. The nature of these responses may determine the susceptibility of the tissue and its ability to resolve the infection.ResultsTranscriptional profiling was employed to measure changes in gene expression occurring in bovine mammary tissues sampled from three dairy cows after brief and graded intramammary challenges with S. aureus. These limited challenges had no significant effect on the expression pattern of the gene encoding β-casein but caused coordinated up-regulation of a number of cytokines and chemokines involved in pro-inflammatory responses. In addition, the enhanced expression of two genes, S100 calcium-binding protein A12 (S100A12) and Pentraxin-3 (PTX3) corresponded with significantly increased levels of their proteins in milk from infected udders. Both genes were shown to be expressed by mammary epithelial cells grown in culture after stimulation with lipopolysaccharide. There was also a strong correlation between somatic cell count, a widely used measure of mastitis, and the level of S100A12 in milk from a herd of dairy cows. Recombinant S100A12 inhibited growth of Escherichia coli in vitro and recombinant PTX3 bound to E. coli as well as C1q, a subunit of the first component of the complement cascade.ConclusionThe transcriptional responses in infected bovine mammary tissue, even at low doses of bacteria and short periods of infection, probably reflect the combined contributions of gene expression changes resulting from the activation of mammary epithelial cells and infiltrating immune cells. The secretion of a number of proinflammatory cytokines and chemokines from mammary epithelial cells stimulated by the bacteria serves to trigger the recruitment and activation of neutrophils in mammary tissue. The presence of S100A12 and PTX3 in milk from infected udder quarters may increase the anti-bacterial properties of milk thereby helping to resolve the mammary tissue infection as well as potentially contributing to the maturation of the newborn calf epithelium and establishment of the newborn gut microbial population.

Lucilia cuprina: Inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane

A culture system has been established to produce gram amounts of peritrophic membrane from larvae of the sheep blowfly, Lucilia cuprina. Peritrophic membrane obtained from this culture has been used to immunize sheep. The immunization produced an immune response which resulted in the average weight of larvae on immunized sheep being only 50% of that of larvae grown on control sheep (P < 0.05). Fractionation of the components of the peritrophic membrane followed by immunization trials showed that the protective antigen fraction comprised material that could only be solubilized by harsh agents such as 4 M-urea. Even after solubilization by 4 M-urea, the protective antigens were able to produce a protective immune response which reduced growth of larvae on immunized sheep to 55% of larvae grown on control sheep (P < 0.05). This immune response which reduced growth of the larvae did not cause gross morphological damage to the larvae.

The development of photochemically crosslinked native fibrinogen as a rapidly formed and mechanically strong surgical tissue sealant.

We recently reported the generation of a highly elastic, crosslinked protein biomaterial via a rapid photochemical process using visible light illumination. In light of these findings, we predicted that other unmodified, tyrosine-rich, self-associating proteins might also be susceptible to this covalent crosslinking method. Here we show that unmodified native fibrinogen can also be photochemically crosslinked into an elastic hydrogel biomaterial through the rapid formation of intermolecular dityrosine. Photochemically crosslinked fibrinogen forms tissue sealant bonds at least 5-fold stronger than commercial fibrin glue and is capable of producing maximum bond strength within 20s. In vitro studies showed that components of the photochemical crosslinking reaction are non-toxic to cells. This material will find useful application in various surgical procedures where rapid curing for high strength tissue sealing is required.

Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro.

The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro. One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant. The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media. These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.

CDNA AND DEDUCED AMINO ACID SEQUENCES OF A PERITROPHIC MEMBRANE GLYCOPROTEIN, 'PERITROPHIN-48', FROM THE LARVAE OF LUCILIA CUPRINA

The gut of most insects is lined with a semi-permeable peritrophic membrane (or peritrophic matrix) composed of chitin, proteoglycans and proteins. Despite the probable importance of the peritrophic membrane in facilitating the digestive process and protecting insects from invasion by micro-organisms and parasites, there has been little characterization of the specific components and their interactions within this acellular structure. Here we report the characterization of an integral peritrophic membrane glycoprotein, peritrophin-48, from the larvae of the fly Lucilia cuprina, a primary agent of cutaneous myiasis in sheep. Peritrophin-48 was purified from peritrophic membrane obtained by larval culture and its location within the peritrophic membrane determined by immuno-fluorescence and immuno-gold localizations. The cDNA coding for peritrophin-48 was cloned and sequenced. The deduced amino acid sequence codes for a protein of 375 amino acids containing an amino-terminal signal sequence followed by five similar, but non-identical domains, each approximately 65-70 amino acids in length and characterised by a specific register of six cysteines. The deduced amino acid sequence shows significant similarity to two other peritrophic membrane proteins, peritrophin-95 and peritrophin-44, from the same species. A reverse transcriptase-PCR approach indicated that there are several highly related peritrophin-48 genes expressed in each individual. Reverse transcriptase-PCR also demonstrated the expression of peritrophin-48 in all three larval instars and adults but not pupae or eggs. Peritrophin-48 was expressed only by the cardia and by the larval midgut. A simple structural model of a basic unit of a type 2 peritrophic membrane is presented.

The major excretory/secretory protease from Lucilia cuprina larvae is also a gut digestive protease

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.