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Isolation and structure elucidation of natural products from marine sponges and an invertebrate were performed. The marine sponges and invertebrate were obtained from three locations including South East Queensland, in Australia, Pontianak in West Kalimantan, and Tulamben, in Bali, Indonesia. The natural products were purified using chromatographic techniques, the structures were elucidated by means of extensive 1D and 2D NMR spectroscopy and some were confirmed by X-ray crystallography. Five known compounds and potentially a new metabolite were identified from three different sponges and a species of nudibranch obtained in Mooloolaba, South East Queensland in Australia. Furospinosulin-1 (4.5), a linear sesterterpenoid, was identified from a sponge coded 20-1-07-1-7 while imidazole alkaloids, preclathridine A (4.9) and clathridine (4.10), were characterized from sponge 22-4-07-2-1. The ethyl acetate extract of sponge 14-7-07-1-1 yielded a polyacetylene fulvinol-like compound (4.15) and potentially a new metabolite compound 5 (4.21). Additionally, a specimen of Chromodoris kuiteri furnished a cyclic macrolide latrunculin A (4.27). There were six secondary metabolites identified from Aaptos aaptos, two of which to the best of our knowledge were new compounds. Aaptamine (5.1), a chemotaxonomic marker of the sponge A. aaptos, 9-demethylaaptamine (5.3) and a biosynthetically unrelated compound (-)-jaspamide (5.31) were found as major constituents of the dichloromethane extract of the sponge. On the other hand, a known indole-3-carbaldehyde (5.10), and the two new natural products methyl 3-(8,9-dimethoxy-4H-benzo[de][1,6]naphthyridin-4-yl)propanoate (5.11) and 8,9-dimethoxy-4H-benzo[de][1,6]naphthyridine-5,6-dione (5.30) were isolated as minor components. During a two-month fieldwork trip to Tulamben, Bali, six different sponges were obtained. Investigation of a blue colored sponge Petrosia sp. afforded two isoquinolinequinone metabolites, namely mimosamycin (5.35) and O-demethylrenierone (5.36). A new 3-alkylpiperidine metabolite, tetradehydrohaliclonacyclamine A (6.28), was isolated from a sponge Halichondria sp. The structure and relative stereochemistry of 6.28 were determined from analysis of 2D NMR data and interpretation of coupling constants. Suitable crystals that were grown from hexane: ethyl acetate (1:3) allowed the determination of the absolute configuration of 6.28 and it was established to be 2S, 3S, 7S, and 9S on the basis of X-ray crystallographic data. The isolated compound (6.28) appeared to be as a single enantiomer according to chiral HPLC. The parent compounds, haliclonacyclamine A (6.19) and B (6.20), were re-isolated from a sample (coded BK-Hal-12-AIK) obtained from a previous project on Haliclona in our group. Their absolute configurations were determined for the first time by X-ray crystallography and they were established to be 2R, 3R, 7R, and 9R.

Isolation and structure elucidation of natural products from marine sponges and an invertebrate were performed. The marine sponges and invertebrate were obtained from three locations including South East Queensland, in Australia, Pontianak in West Kalimantan, and Tulamben, in Bali, Indonesia. The natural products were purified using chromatographic techniques, the structures were elucidated by means of extensive 1D and 2D NMR spectroscopy and some were confirmed by X-ray crystallography. Five known compounds and potentially a new metabolite were identified from three different sponges and a species of nudibranch obtained in Mooloolaba, South East Queensland in Australia. Furospinosulin-1 (4.5), a linear sesterterpenoid, was identified from a sponge coded 20-1-07-1-7 while imidazole alkaloids, preclathridine A (4.9) and clathridine (4.10), were characterized from sponge 22-4-07-2-1. The ethyl acetate extract of sponge 14-7-07-1-1 yielded a polyacetylene fulvinol-like compound (4.15) and potentially a new metabolite compound 5 (4.21). Additionally, a specimen of Chromodoris kuiteri furnished a cyclic macrolide latrunculin A (4.27). There were six secondary metabolites identified from Aaptos aaptos, two of which to the best of our knowledge were new compounds. Aaptamine (5.1), a chemotaxonomic marker of the sponge A. aaptos, 9-demethylaaptamine (5.3) and a biosynthetically unrelated compound (-)-jaspamide (5.31) were found as major constituents of the dichloromethane extract of the sponge. On the other hand, a known indole-3-carbaldehyde (5.10), and the two new natural products methyl 3-(8,9-dimethoxy-4H-benzo[de][1,6]naphthyridin-4-yl)propanoate (5.11) and 8,9-dimethoxy-4H-benzo[de][1,6]naphthyridine-5,6-dione (5.30) were isolated as minor components. During a two-month fieldwork trip to Tulamben, Bali, six different sponges were obtained. Investigation of a blue colored sponge Petrosia sp. afforded two isoquinolinequinone metabolites, namely mimosamycin (5.35) and O-demethylrenierone (5.36). A new 3-alkylpiperidine metabolite, tetradehydrohaliclonacyclamine A (6.28), was isolated from a sponge Halichondria sp. The structure and relative stereochemistry of 6.28 were determined from analysis of 2D NMR data and interpretation of coupling constants. Suitable crystals that were grown from hexane: ethyl acetate (1:3) allowed the determination of the absolute configuration of 6.28 and it was established to be 2S, 3S, 7S, and 9S on the basis of X-ray crystallographic data. The isolated compound (6.28) appeared to be as a single enantiomer according to chiral HPLC. The parent compounds, haliclonacyclamine A (6.19) and B (6.20), were re-isolated from a sample (coded BK-Hal-12-AIK) obtained from a previous project on Haliclona in our group. Their absolute configurations were determined for the first time by X-ray crystallography and they were established to be 2R, 3R, 7R, and 9R.

The Australian paralysis tick, Ixodes holocyclus causes a potentially fatal paralysis in domestic animals, livestock and humans with companion animals (mainly dogs) most commonly affected. Current treatment regimes include administration of a commercial tick anti-serum (TAS), prepared as hyperimmune serum in dogs, to neutralise the effects of the toxin. However, each new batch must be standardised using an expensive and highly subjective bioassay performed in neonatal mice. There is currently an urgent need for a more cost effective and rapid in vitro assay that can be more objectively and accurately quantified. Further understanding of the composition of the toxin molecule is also required to develop toxin-specific reagents necessary for these assays. One of the main objectives of this study was to develop a suitable immunoassay to replace the existing mouse bioassay for assessing batches of tick anti-sera for use in tick paralysis therapy in dogs. Initially an enzyme-linked immunosorbent assay (ELISA) was established to detect and quantify antibody specific for I. holocyclus toxin in dog sera. Using a partially purified antigen extracted from I. holocyclus salivary glands, good discrimination was achieved between reactive (hyperimmune) and non-reactive (naïve) sera. The hyperimmune dog sera reacted very strongly with the antigen compared to negligible reactions of serum from dogs not exposed to I. holocyclus. The reactions of hyperimmune sera were also significantly weaker to a non-toxin antigen control extracted from the salivary glands of the non-toxic tick Rhipicephalus microplus, indicating the assay was detecting toxin-specific responses. Furthermore, each of the hyperimmune sera that reacted strongly and specifically with the I. holocyclus antigen in the ELISA also strongly neutralised toxin in the mouse bioassay. Together these findings support the suitability of this ELISA for assessing the potency of batches of commercial dog hyperimmune sera for use as therapy for tick paralysis in dogs. Sera from dogs that were experimentally infested with ticks and sera from patient dogs, presenting at veterinary clinics with signs of tick paralysis, were also screened for antibodies to I. holocyclus antigen using the ELISA. Twenty-eight out of 29 sera from animals with single or multiple exposures to ticks failed to recognise the I. holocyclus antigen indicating the ELISA is not suitable as a diagnostic test to detect toxin-specific antibodies in animals with limited exposure to I. holocyclus infestation. A panel of toxin-specific monoclonal antibodies (mAbs) was produced as research tools to analyse and purify tick toxin components. Rats were successfully immunised against tick toxin using a combination of inoculation of partially purified salivary gland antigen and exposure to tick infestation. The latter approach preserved the native confirmation of the toxin using a natural route of immunisation and rats were chosen due to their high tolerance of multiple tick infestations over several days. While fusion of rat spleen cells with mouse myeloma cells has been reported several times in the literature, the resulting hybridomas are unstable with fastidious culture requirements. Optimisation of the culture conditions revealed that most rat-mouse hybridoma lines grew best in serum-free medium supplemented with 5% foetal bovine serum. Of 600 hybridomas produced, only 12 were shown to be specific for the Ixodes antigen, as determined by ELISA. A selection of these hybridomas representing various patterns of affinity and/or antigen specificity were further analysed for toxin-neutralising ability in a mouse bioassay. Notably, the most potent toxin-neutralising mAb in mice, showed a specific but relatively moderate reaction to Ixodes antigen in the ELISA. The most potent toxin-neutralising mAbs inactivated toxin as strongly as the commercial TAS used for immunotherapy in dogs with tick paralysis. This suggests that mAbs may present an alternative source of immunotherapy, providing a potentially endless supply of a highly consistent reagent and negating the need to use live animals for both the production of tick antiserum and the continual testing of reagent batches. The toxin-neutralising mAbs were also used to analyse I. holocyclus toxin in polyacrylamide gel electrophoresis (PAGE) and Western blot to identify specific toxin proteins. The most potent neutralising mAbs consistently recognised high MW proteins (100-200 kDa) in a smeared pattern. Although this was contrary to previous reports of low molecular weight components (3-5 kDa) in holocyclotoxin, this study was the first to use mAbs prepared to native toxin. The large molecular weight structures likely represent presucursors to, or complexes of the smaller peptides, previously identified. When the Toxin-neutralising mAbs were assessed as ligands to affinity purify toxin components from crude Ixodes SG extracts, toxin components of 110 and 32 kDa were consistently identified. These purified proteins represent good candidates for N-terminal sequencing to further identify the toxin components in I.holocyclus salivary glands.

The Australian paralysis tick, Ixodes holocyclus causes a potentially fatal paralysis in domestic animals, livestock and humans with companion animals (mainly dogs) most commonly affected. Current treatment regimes include administration of a commercial tick anti-serum (TAS), prepared as hyperimmune serum in dogs, to neutralise the effects of the toxin. However, each new batch must be standardised using an expensive and highly subjective bioassay performed in neonatal mice. There is currently an urgent need for a more cost effective and rapid in vitro assay that can be more objectively and accurately quantified. Further understanding of the composition of the toxin molecule is also required to develop toxin-specific reagents necessary for these assays. One of the main objectives of this study was to develop a suitable immunoassay to replace the existing mouse bioassay for assessing batches of tick anti-sera for use in tick paralysis therapy in dogs. Initially an enzyme-linked immunosorbent assay (ELISA) was established to detect and quantify antibody specific for I. holocyclus toxin in dog sera. Using a partially purified antigen extracted from I. holocyclus salivary glands, good discrimination was achieved between reactive (hyperimmune) and non-reactive (naïve) sera. The hyperimmune dog sera reacted very strongly with the antigen compared to negligible reactions of serum from dogs not exposed to I. holocyclus. The reactions of hyperimmune sera were also significantly weaker to a non-toxin antigen control extracted from the salivary glands of the non-toxic tick Rhipicephalus microplus, indicating the assay was detecting toxin-specific responses. Furthermore, each of the hyperimmune sera that reacted strongly and specifically with the I. holocyclus antigen in the ELISA also strongly neutralised toxin in the mouse bioassay. Together these findings support the suitability of this ELISA for assessing the potency of batches of commercial dog hyperimmune sera for use as therapy for tick paralysis in dogs. Sera from dogs that were experimentally infested with ticks and sera from patient dogs, presenting at veterinary clinics with signs of tick paralysis, were also screened for antibodies to I. holocyclus antigen using the ELISA. Twenty-eight out of 29 sera from animals with single or multiple exposures to ticks failed to recognise the I. holocyclus antigen indicating the ELISA is not suitable as a diagnostic test to detect toxin-specific antibodies in animals with limited exposure to I. holocyclus infestation. A panel of toxin-specific monoclonal antibodies (mAbs) was produced as research tools to analyse and purify tick toxin components. Rats were successfully immunised against tick toxin using a combination of inoculation of partially purified salivary gland antigen and exposure to tick infestation. The latter approach preserved the native confirmation of the toxin using a natural route of immunisation and rats were chosen due to their high tolerance of multiple tick infestations over several days. While fusion of rat spleen cells with mouse myeloma cells has been reported several times in the literature, the resulting hybridomas are unstable with fastidious culture requirements. Optimisation of the culture conditions revealed that most rat-mouse hybridoma lines grew best in serum-free medium supplemented with 5% foetal bovine serum. Of 600 hybridomas produced, only 12 were shown to be specific for the Ixodes antigen, as determined by ELISA. A selection of these hybridomas representing various patterns of affinity and/or antigen specificity were further analysed for toxin-neutralising ability in a mouse bioassay. Notably, the most potent toxin-neutralising mAb in mice, showed a specific but relatively moderate reaction to Ixodes antigen in the ELISA. The most potent toxin-neutralising mAbs inactivated toxin as strongly as the commercial TAS used for immunotherapy in dogs with tick paralysis. This suggests that mAbs may present an alternative source of immunotherapy, providing a potentially endless supply of a highly consistent reagent and negating the need to use live animals for both the production of tick antiserum and the continual testing of reagent batches. The toxin-neutralising mAbs were also used to analyse I. holocyclus toxin in polyacrylamide gel electrophoresis (PAGE) and Western blot to identify specific toxin proteins. The most potent neutralising mAbs consistently recognised high MW proteins (100-200 kDa) in a smeared pattern. Although this was contrary to previous reports of low molecular weight components (3-5 kDa) in holocyclotoxin, this study was the first to use mAbs prepared to native toxin. The large molecular weight structures likely represent presucursors to, or complexes of the smaller peptides, previously identified. When the Toxin-neutralising mAbs were assessed as ligands to affinity purify toxin components from crude Ixodes SG extracts, toxin components of 110 and 32 kDa were consistently identified. These purified proteins represent good candidates for N-terminal sequencing to further identify the toxin components in I.holocyclus salivary glands.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of proteins intitially characterised in the late 1980’s by their presence in complexes that form on nascent RNA transcripts. This definition was purely operational, and was based on protein isolation techniques available at that time. Since then, the tendency to refer to and view the hnRNPs as a protein family has become increasingly prevalent, although there has been no systematic sequence- or structure-based study of their evolutionary history. While the hnRNPs share some structural characteristics (modular structure, presence of RNA-binding domains) and functional properties (binding to RNA, involvement in multiple steps of RNA processing), these criteria also apply to other types of RNA binding proteins (RBPs), such as the SR and ELAV families of proteins. Thus, we have adopted a more methodical and rigorous approach to the classification of hnRNPs and other RBPs, through the phylogenetic analysis of their sequences and domains. Besides establishing phylogenetic relationships and simplifying nomenclature, studying the evolutionary divergence of the hnRNPs is important for understanding their functional features. The hnRNP A/B subfamily is comprised of paralogues A1, A2/B1, A3 and A0, which exhibit a high level of similarity at both the sequence and structural level. While they are often treated as functional homologues, they are not functionally identical. Hence, we undertook a detailed comparison of their sequences, and found that the introduction of novel splicing signals or mutation of existing sequence elements has led to changes in alternative splicing patterns between the paralogues, which may affect the regulation of their expression and their RNA binding properties. In addition, we also investigated species-specific alternative splicing of the hnRNPs A/B, which has significant implications for the interpretation of current research, since different research groups tend to use different model organisms in their experiments. Hence, exploration of the sequence divergence of the hnRNPs A/B has provided some clues as to how their functional differences arose, and also highlighted the need to take species-specific splicing into consideration. Alternative splicing can create functional variation not only between paralogues, but also between splice variants. hnRNP A2/B1, which has a well-established role in mRNA trafficking in neuronal cells, has four spliceoforms. In order to study the contribution of each isoform to this process, we investigated isoform-specific variations in intracellular localisation, and expression in different developmental stages and species. We found that in rat, minor isoform A2b was the predominant isoform in the cytoplasm, and may be the key player in mRNA trafficking. These findings demonstrate the importance of considering individual isoforms (including those expressed in low abundance) when studying the function of alternatively spliced proteins, especially when the function is restricted to a particular subcellular compartment. In addition to its cytoplasmic role in mRNA trafficking, hnRNP A2/B1, and the other hnRNPs A/B, have multiple nuclear functions, including packaging of nascent transcripts, nuclear export of mRNA, regulation of alternative splicing and telomere maintenance. These processes take place in discrete regions within the nucleus, and thus we examined the subnuclear distribution of the hnRNPs A/B. We found that hnRNP A1 had a localisation pattern distinct from that of A2/B1 and A3, and that these patterns were spatially and temporally regulated. Hence, the evolutionary divergence of the hnRNPs A/B has affected the localisation, expression and splicing patterns of these proteins, which we have examined at multiple levels, including comparisons across all hnRNPs, within the hnRNP A/B paralogues, and between the hnRNP A2/B1 splice variants. As the hnRNPs A/B are involved in almost every step in RNA processing, this functional diversity has significant implications for transcriptomic complexity. Furthermore, our findings highlight the importance of taking species- and isoform-specific differences into account when investigating protein function. In conclusion, this study of the hnRNPs A/B provides a conceptual framework for exploring the relationships between sequence, structural and functional divergence, which may be applicable to protein families in general.