Comparative Biochemistry and Physiology B, Dec 1, 2007
Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in othe... more Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca2+ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (Km=4.45+/-1.46 mM) than for the diphenol, l-DOPA (Km=10.27+/-1.33 mM). Maximum enzyme activity was evident at 37 degrees C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.
Complement activation in vertebrates has traditionally been associated with the classical (antibo... more Complement activation in vertebrates has traditionally been associated with the classical (antibody mediated) or alternative (spontaneously hydrolytic) pathways. Recently, a new mechanism of complement activation has been identified in which a collagenous lectin (collectin), mannose binding protein (MBP) and its associated serine protease (MASP) activate the central component of the complement system, C3 (Epstein, et al., 1996, Holmskov, et al., 1994, Hoppe and Reid, 1994, Lu, 1997). Two components of this lectin-mediated complement pathway have already been identified in invertebrates. Echinoderms express a C3 homologue and a number of other complement components, whilst a MASP-like serine protease and a C3 homologue have been identified in tunicates (Al-Sharif, et al., 1998, Azumi, et al., 1993, Nonaka, et al., 1998, Smith, et al., 1996, Smith, et al., 1998).
Developmental and Comparative Immunology, May 1, 2013
The purple sea urchin has a complex immune system that is likely mediated by gene expression in c... more The purple sea urchin has a complex immune system that is likely mediated by gene expression in coelomocytes (blood cells). A broad array of potential immune receptors and immune response proteins has been deduced from their gene models. Here we use shotgun mass spectrometry to describe 307 proteins with possible immune function in sea urchins including proteins involved in the complement pathway and numerous SRCRs. The relative abundance of dual oxidase 1, ceruloplasmin, ferritin and transferrin suggests the production of reactive oxygen species in coelomocytes and the sequestration of iron. Proteins such as selectin, cadherin, talin, galectin, amassin and the Von Willebrand factor may be involved in generating a strong clotting reaction. Cell signaling proteins include a guanine nucleotide binding protein, the Rho GDP dissociation factor, calcium storage molecules and a variety of lipoproteins. However, based on this dataset, the expression of TLRs, NLRs and fibrinogen domain containing proteins in coelomic fluid and coelomocytes could not be verified.
Viral diseases are a significant cause of mortality and morbidity in oysters. Economic losses ste... more Viral diseases are a significant cause of mortality and morbidity in oysters. Economic losses stemming from herpesvirus infection of farmed oysters results in significant economic loss to the aquaculture industry. In Eastern Australia, farmed Pacific oysters (Crassostrea gigas, PO) are susceptible to infection by ostreid herpesviruses, while the Sydney Rock oysters (Saccostrea glomerata, SRO) appears to be unaffected by this virus. In this study, we investigated the proteomic responses of these two species of oysters to generic double stranded RNAs (poly I:C and poly A:U). Analysis of proteomic data (iTRAQ) indicated that there were significant differences in the proteomic responses of the two oyster species resulting from this treatment. A Fisher exact test was undertaken to determine if specific gene ontology terms were enriched in the proteomic datasets. We observed that several biological processes, cellular component and molecular function were unique to the different datasets. For example, a number of proteins implicated in the TLR signalling pathway were associated with the SRO dataset, but were absent in the PO dataset. The RNA interference pathway protein, Piwi, was also identified in the SRO dataset, but not in the other. These results suggest that the differences in the proteomic responses to dsRNA may underpin the biological differences in viral susceptibility exhibited by the two oyster species. Previous studies had evaluated PO transcriptional responses to dsRNA or ostreid herpesvirus DNA. The vast majority of the targets that were identified in those studies were not present in our proteomic datasets, although they were present in the RNA extracted from the very same tissues. Taken together, our data indicate that there are substantial disparities between transcriptomic and proteomic responses to dsRNA challenge and a comprehensive account of the oysters' biological responses to these treatments must take into account that disparity.
The sea urchin, Heliocidaris erythrogramma, has potent antibacterial activity in its coelomic flu... more The sea urchin, Heliocidaris erythrogramma, has potent antibacterial activity in its coelomic fluid. Antibacterial activity was detected by a colorimetric bactericidal assay, which revealed a minimum inhibitory concentration of 680 ppm coelomic fluid v/v in FSW. The antibacterial activity of a number of molecules was induced by bacterial challenge with maximal antibacterial activity occurring after 12 h. The molecules responsible for antibacterial activity were heat-stable and also induced by non-specific challenge. Polar extracts of coelomic fluid indicated that at least one of the factors contributing to antibacterial activity was proteinaceous. The antimicrobial peptide was approximately 13 9 kDa as determined by SDS-PAGE. The amino acid sequence and potency of the antimicrobial peptide is also discussed.1 page(s
There is limited information on bacterial communities attached to marine surfaces. These surface ... more There is limited information on bacterial communities attached to marine surfaces. These surface attached bacterial communities can vary at a micro scale and these differences may be due to surface characteristics in marine environments. The current study investigates the heterogeneity of bacterial communities on five different marine invertebrates (Heliocidaris erythrogramma, Austrocochlea concamerata, Crassostrea gigas, Dendrilla rosea, and Actinia tenebrosa), the alga, Lobophora variegata and marine gravel from a 20 m × 20 m quadrant in Camp Cove, Sydney Harbour, Australia. Terminal restriction fragment length polymorphism (TRFLP) of 16S sequences showed that each surface contained unique combinations of TRFLP fragment lengths. Phylogenetic analysis of random clones picked from clone libraries constructed from the amplified 16S sequences revealed that 16S sequences from the communities on different surfaces clustered into distinct clades. None of the bacteria identified by 16S sequencing of the whole (uncultured) microbial communities was detected after cultivation. Overall, the study shows surface type plays a major role in shaping microbial communities in marine environments.
Sydney rock oysters (Saccostrea glomerata) were exposed to environmental stressors at contaminate... more Sydney rock oysters (Saccostrea glomerata) were exposed to environmental stressors at contaminated field sites or in a controlled laboratory setting. RNA seq transcriptome data were generated for the gill and digestive gland using Roche's 454 pyrosequencing technology. 28,685 contigs were de novo assembled which encoded 11,671 different protein products. The data will act as a reference for future studies in ecology, immunology and environmental toxicology.
Progress in molecular and subcellular biology, 2003
Tunicates (ascidians or sea squirts) are a large group of invertebrate chordates that are closely... more Tunicates (ascidians or sea squirts) are a large group of invertebrate chordates that are closely related to vertebrates. Their critical phylogenetic position has stimulated substantial interest in their host defense ("immune") responses. Whilst this interest has generated a wealth of knowledge regarding the humoral and cellular mechanisms that undertake defensive responses, there is less known about the regulation of those reactions. This chapter focuses on three cellular responses (cell proliferation, phagocytosis and chemotaxis) that are known to be regulated by cytophilic humoral molecules. Some of the humoral factors that affect these responses have functional and physicochemical similarities to vertebrate cytokines, like interleukin-1. However, the only regulatory molecules that have been characterized at a molecular level bear far greater similarity to C-type lectins or complement components.
Comparative Biochemistry and Physiology B, Dec 1, 2007
Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in othe... more Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca2+ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (Km=4.45+/-1.46 mM) than for the diphenol, l-DOPA (Km=10.27+/-1.33 mM). Maximum enzyme activity was evident at 37 degrees C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.
Complement activation in vertebrates has traditionally been associated with the classical (antibo... more Complement activation in vertebrates has traditionally been associated with the classical (antibody mediated) or alternative (spontaneously hydrolytic) pathways. Recently, a new mechanism of complement activation has been identified in which a collagenous lectin (collectin), mannose binding protein (MBP) and its associated serine protease (MASP) activate the central component of the complement system, C3 (Epstein, et al., 1996, Holmskov, et al., 1994, Hoppe and Reid, 1994, Lu, 1997). Two components of this lectin-mediated complement pathway have already been identified in invertebrates. Echinoderms express a C3 homologue and a number of other complement components, whilst a MASP-like serine protease and a C3 homologue have been identified in tunicates (Al-Sharif, et al., 1998, Azumi, et al., 1993, Nonaka, et al., 1998, Smith, et al., 1996, Smith, et al., 1998).
Developmental and Comparative Immunology, May 1, 2013
The purple sea urchin has a complex immune system that is likely mediated by gene expression in c... more The purple sea urchin has a complex immune system that is likely mediated by gene expression in coelomocytes (blood cells). A broad array of potential immune receptors and immune response proteins has been deduced from their gene models. Here we use shotgun mass spectrometry to describe 307 proteins with possible immune function in sea urchins including proteins involved in the complement pathway and numerous SRCRs. The relative abundance of dual oxidase 1, ceruloplasmin, ferritin and transferrin suggests the production of reactive oxygen species in coelomocytes and the sequestration of iron. Proteins such as selectin, cadherin, talin, galectin, amassin and the Von Willebrand factor may be involved in generating a strong clotting reaction. Cell signaling proteins include a guanine nucleotide binding protein, the Rho GDP dissociation factor, calcium storage molecules and a variety of lipoproteins. However, based on this dataset, the expression of TLRs, NLRs and fibrinogen domain containing proteins in coelomic fluid and coelomocytes could not be verified.
Viral diseases are a significant cause of mortality and morbidity in oysters. Economic losses ste... more Viral diseases are a significant cause of mortality and morbidity in oysters. Economic losses stemming from herpesvirus infection of farmed oysters results in significant economic loss to the aquaculture industry. In Eastern Australia, farmed Pacific oysters (Crassostrea gigas, PO) are susceptible to infection by ostreid herpesviruses, while the Sydney Rock oysters (Saccostrea glomerata, SRO) appears to be unaffected by this virus. In this study, we investigated the proteomic responses of these two species of oysters to generic double stranded RNAs (poly I:C and poly A:U). Analysis of proteomic data (iTRAQ) indicated that there were significant differences in the proteomic responses of the two oyster species resulting from this treatment. A Fisher exact test was undertaken to determine if specific gene ontology terms were enriched in the proteomic datasets. We observed that several biological processes, cellular component and molecular function were unique to the different datasets. For example, a number of proteins implicated in the TLR signalling pathway were associated with the SRO dataset, but were absent in the PO dataset. The RNA interference pathway protein, Piwi, was also identified in the SRO dataset, but not in the other. These results suggest that the differences in the proteomic responses to dsRNA may underpin the biological differences in viral susceptibility exhibited by the two oyster species. Previous studies had evaluated PO transcriptional responses to dsRNA or ostreid herpesvirus DNA. The vast majority of the targets that were identified in those studies were not present in our proteomic datasets, although they were present in the RNA extracted from the very same tissues. Taken together, our data indicate that there are substantial disparities between transcriptomic and proteomic responses to dsRNA challenge and a comprehensive account of the oysters' biological responses to these treatments must take into account that disparity.
The sea urchin, Heliocidaris erythrogramma, has potent antibacterial activity in its coelomic flu... more The sea urchin, Heliocidaris erythrogramma, has potent antibacterial activity in its coelomic fluid. Antibacterial activity was detected by a colorimetric bactericidal assay, which revealed a minimum inhibitory concentration of 680 ppm coelomic fluid v/v in FSW. The antibacterial activity of a number of molecules was induced by bacterial challenge with maximal antibacterial activity occurring after 12 h. The molecules responsible for antibacterial activity were heat-stable and also induced by non-specific challenge. Polar extracts of coelomic fluid indicated that at least one of the factors contributing to antibacterial activity was proteinaceous. The antimicrobial peptide was approximately 13 9 kDa as determined by SDS-PAGE. The amino acid sequence and potency of the antimicrobial peptide is also discussed.1 page(s
There is limited information on bacterial communities attached to marine surfaces. These surface ... more There is limited information on bacterial communities attached to marine surfaces. These surface attached bacterial communities can vary at a micro scale and these differences may be due to surface characteristics in marine environments. The current study investigates the heterogeneity of bacterial communities on five different marine invertebrates (Heliocidaris erythrogramma, Austrocochlea concamerata, Crassostrea gigas, Dendrilla rosea, and Actinia tenebrosa), the alga, Lobophora variegata and marine gravel from a 20 m × 20 m quadrant in Camp Cove, Sydney Harbour, Australia. Terminal restriction fragment length polymorphism (TRFLP) of 16S sequences showed that each surface contained unique combinations of TRFLP fragment lengths. Phylogenetic analysis of random clones picked from clone libraries constructed from the amplified 16S sequences revealed that 16S sequences from the communities on different surfaces clustered into distinct clades. None of the bacteria identified by 16S sequencing of the whole (uncultured) microbial communities was detected after cultivation. Overall, the study shows surface type plays a major role in shaping microbial communities in marine environments.
Sydney rock oysters (Saccostrea glomerata) were exposed to environmental stressors at contaminate... more Sydney rock oysters (Saccostrea glomerata) were exposed to environmental stressors at contaminated field sites or in a controlled laboratory setting. RNA seq transcriptome data were generated for the gill and digestive gland using Roche's 454 pyrosequencing technology. 28,685 contigs were de novo assembled which encoded 11,671 different protein products. The data will act as a reference for future studies in ecology, immunology and environmental toxicology.
Progress in molecular and subcellular biology, 2003
Tunicates (ascidians or sea squirts) are a large group of invertebrate chordates that are closely... more Tunicates (ascidians or sea squirts) are a large group of invertebrate chordates that are closely related to vertebrates. Their critical phylogenetic position has stimulated substantial interest in their host defense ("immune") responses. Whilst this interest has generated a wealth of knowledge regarding the humoral and cellular mechanisms that undertake defensive responses, there is less known about the regulation of those reactions. This chapter focuses on three cellular responses (cell proliferation, phagocytosis and chemotaxis) that are known to be regulated by cytophilic humoral molecules. Some of the humoral factors that affect these responses have functional and physicochemical similarities to vertebrate cytokines, like interleukin-1. However, the only regulatory molecules that have been characterized at a molecular level bear far greater similarity to C-type lectins or complement components.
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