17 results for Puddick, Jonathan

  • A comparative proteomics approach to studying skeletal muscle mitochondria from myostatin knockout mice

    Puddick, Jonathan (2006)

    Masters thesis
    University of Waikato

    Myostatin is a negative regulator of muscle growth. When it is not present or non-functional double-muscling occurs, the primary characteristic of this phenotype being an increase in muscle mass. Another characteristic of double-muscling is an increased proportion of type IIB muscle fibres, which rely on glycolysis as their primary energy source, as opposed to type IIA and type I fibres which rely on oxidative phosphorylation. This switch in muscle metabolism directly impacts on the mitochondria, as mitochondria from glycolytic muscle fibres have been shown to have differences in metabolic activity. The increased proportion of glycolytic muscle fibres present in myostatin knockout animals provides a unique model to investigate alterations in muscle fibre type metabolism. The mouse model of myostatin knockout utilised during this study was generated by genetic deletion of exon three of the myostatin gene. Verification of this knockout was attempted by western blot analysis, but only the latency associated protein (LAP) was detected. Interestingly, the LAP was barely detectable in the knockout muscle suggesting deletion of exon three affects binding of anti-myostatin antibodies to the LAP, as that part of the gene is not deleted. A comparison of the basal mitochondrial stress levels was made, also by western blot analysis. The knockout mitochondria showed no change in levels of heat shock protein 60 or superoxide dismutase 2, indicating that they are not being subjected to any increased stress due to the myostatin knockout phenotype. A comparative proteomics approach was used to detect changes in the mitochondrial proteome of myostatin knockout gastrocnemius muscle to gain clues to how mitochondria from glycolytic muscle fibres differ from those present in oxidative fibres. This was undertaken using two-dimensional electrophoresis (2-DE), in-gel tryptic digests and peptide mass fingerprinting by mass spectrometry. A 2-DE gel protein loading of 220 g was shown to give the best protein spot resolution and the most crucial step in the loading process was found to be the laying of the immobilized pH gradient, which had to be performed very carefully to obtain a consistent loading pattern. This study resolved only around 160 protein spots out of the estimated 1,000 to 2,000 proteins present in the mitochondria. Modulation of six proteins was seen at a plt0.1 level, but were unable to be identified using the current methodology. More abundant mitochondrial proteins were able to be identified, but showed no significant modulation. Malate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase, which were identified during this study, have been reported to have decreased activity in mitochondria from glycolytic muscle fibres. This study suggests that the change in activity observed by other researchers is due to inhibition of these enzymes in the glycolytic fibres or activation in the oxidative fibres.

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  • Proteomes and Signalling Pathways of Antler Stem Cells

    Li, Chunyi; Harper, Anne; Puddick, Jonathan; Wang, Wenying; McMahon, Chris (2012)

    Journal article
    University of Waikato

    As the only known example of complete organ regeneration in mammals, deer antler in the growing or velvet phase is of major interest in developmental biology. This regeneration event initiates from self-renewing antler stem cells that exhibit pluripotency. At present, it remains unclear how the activation and quiescence of antler stem cells are regulated. Therefore, in the present study proteins that were differentially expressed between the antler stem cells and somatic cells (facial periosteum) were identified by a gel-based proteomic technique, and analysed using Ingenuity Pathway Analysis software. Several molecular pathways (PI3K/Akt, ERK/MAPK, p38 MAPK, etc.) were found to be activated during proliferation. Also expressed were the transcription factors POU5F1, SOX2, NANOG and MYC, which are key markers of embryonic stem cells. Expression of these proteins was confirmed in both cultured cells and fresh tissues by Western blot analysis. Therefore, the molecular pathways and transcription factors identified in the current study are common to embryonic and adult stem cells. However, expression of embryonic stem cell transcription factors would suggest that antler stem cells are, potentially, an intermediary stem cell type between embryonic and the more specialized tissue-specific stem cells like those residing in muscle, fat or from a hematopoietic origin. The retention of this embryonic, pluripotent lineage may be of fundamental importance for the subsequent regenerative capacity of antlers.

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  • Spectroscopic Investigations of Oligopeptides from Aquatic Cyanobacteria: Characterisation of New Oligopeptides, Development of Microcystin Quantification Tools and Investigations into Microcystin Production

    Puddick, Jonathan (2013)

    Doctoral thesis
    University of Waikato

    Cyanobacteria (blue-green algae) are a group of ancient prokaryotic organisms which have become synonymous with water quality deterioration. An array of compounds is produced by aquatic cyanobacteria, the largest group being the oligopeptides. A major class of cyanobacterial oligopeptides are the microcystins; cyclic heptapeptides which contain the unique amino acid Adda (3 amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid). Due to their ability to inhibit protein phosphatases 1 and 2A and as they are concentrated in the liver, microcystins can be highly toxic to animals. Anabaenopeptins are cyclic hexapeptides which contain a carbonyl-linked sidechain amino acid and a ring which is cyclised through the sidechain amine of the position two D-lysine. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF MS) analysis of a cyanobacterial bloom sample from Lake Wiritoa (Manawatu, New Zealand) led to the identification of a new anabaenopeptin. The putative structure for anabaenopeptin 906 was constructed using tandem MS (MS/MS) data, in conjunction with the sequences of the known anabaenopeptins. A Microcystis species (CYN06) isolated from Lake Hakanoa (Huntly, New Zealand) was investigated as it produced a large number of microcystin congeners. Liquid chromatography-MS/MS analysis was used to identify seventeen known microcystin congeners and to characterise ten new variants. Two of the new microcystins, MC-FA and MC-WA, were structurally characterised using amino acid analysis and nuclear magnetic resonance (NMR) spectroscopy. The other new congeners included MC FAba, MC WAba, MC FL, MC-WL as well as [Asp³] analogues of MC FA, MC WA, and [Asp³] analogues of two known congeners; MC-RA and MC-RAba. A review of the number of microcystin congeners produced by reported cyanobacterial strains placed CYN06 in the upper ten percentile. However, CYN06 differed from the other strains as it showed relaxed substrate specificity at position two and four and simultaneously produced microcystin congeners which contained no arginine residues, one arginine residue and two arginine residues. Microcystis CYN06 also contained an unidentified microcystin with a mass of 1014.5 Da. Structural characterisation by NMR spectroscopy indicated that it was an analogue of MC-WA which contained N-formylkynurenine at position two. As N-formylkynurenine is a known oxidation product of tryptophan it was proposed that further unidentified microcystins from CYN06 contained two other oxidation products of tryptophan; kynurenine and oxindolyalanine. This is the first report of the presence of oxidised tryptophan residues in microcystins. Analysis of a cyanobacterial hydroterrestrial mat sample collected from Miers Valley in Eastern Antarctica indicated the presence of fourteen new oligopeptides. A combination of MS/MS and amino acid analysis was used to characterise eight new microcystins which each contained a position one glycine. A recently-developed thiol derivatisation technique indicated that the position seven amino acid in each of the microcystins was very likely dehydrobutyrine. Different combinations of variable modifications at positions two, four and five resulted in eight unique structures. These included four microcystins with Adda moieties which possessed an O-acetyl group (ADMAdda) instead of the conventional C9 methoxy. As well as being the first report of microcystins containing a position one glycine, this is the first report of ADMAdda-containing microcystins in the southern hemisphere. The putative structures of six new Antarctic linear peptides were determined through a combination of mass spectrometric techniques. The compounds contained an N terminus with the molecular formula C₉H₁₄NO₂ linked to isoleucine, two aromatic amino acids and an ester linked hydroxyphenyllactic acid. The hydroxyphenyllactic acid C terminus and the unidentified N terminus suggest that these new compounds are a novel class of cyanobacterial oligopeptide. Seven different sample preparation techniques for the quantitative analysis of microcystins by MALDI-TOF MS were assessed for signal reproducibility and sensitivity using a cost-effective internal standard (angiotensin I). The sensitivity of six of the preparations was acceptable, as was the reproducibility for two thin layer preparations performed on a polished steel target. Both thin layer preparations could be used with a MALDI-TOF mass spectrometer which automatically acquires data. The thin-layer-spot preparation could also be used in an automated sample preparation work-flow. Further investigation using this preparation demonstrated that linear quantification of three different microcystin congeners ([Dha⁷] MC-LR, MC-RR and MC-YR) was possible. Use of this MALDI sample preparation will allow large numbers of microcystin-containing samples to be analysed rapidly and at low cost. A batch culture experiment using Microcystis CYN06 exhibited a decreased abundance of arginine-containing microcystins as nitrate concentrations decreased. Linear regression of the relationship between log10 microcystin content and nitrate concentration revealed slopes which were dependent upon the number of arginine residues present in the compound. During this experiment the abundance of congeners with a single arginine residue at position two did not change (p > 0.05), whilst the abundance of the congeners with a position four arginine decreased significantly (p ≤ 0.001). This suggests that there could be an element of selectivity in regards to which arginine in the microcystin structure is modulated and could explain why congener modulation in response to nitrogen concentration has not been observed previously. Whilst it was not proven that nitrogen supply was the causative factor for the congener modulation, the results from this experiment warrant further study in this area. This research has significantly expanded our knowledge of oligopeptide diversity, improved an existing method of quantifying microcystins and shed new light on the regulation of the abundance of microcystin congeners. The identification of eighteen new microcystins is a 16% increase upon the 111 presently characterised. Also reported was the identification of nine oxidised analogues of tryptophan-containing microcystins from Microcystis CYN06. The presence of oxidised Trp residues in microcystins has not been reported previously and will allow researchers working with samples of Trp-containing microcystins to now assign the oxidised analogues. Seven new cyanobacterial oligopeptides were characterised during this study, six of which may belong to a novel class of linear peptides. A sample preparation designed for the quantification of microcystins by MALDI TOF MS gave comparative performance to the previously reported method but was compatible with automated high-throughput sample preparation and data acquisition. For the first time, a culturing experiment showed a relationship between the abundance of arginine-containing microcystins and nitrogen supply.

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  • MALDI-TOF mass spectrometry of Cyanobacteria: a global approach to the discovery of novel secondary metabolites

    Puddick, Jonathan; Prinsep, Michèle R. (2008)

    Journal article
    University of Waikato

    Cyanobacteria (blue-green algae) are a group of ancient prokaryotic organisms dating back between three and four billion years.¹ They have been attributed with oxygenating the earth’s atmosphere² but, since the anthropogenic euthrophication of lakes, ponds and oceans, they have become synonymous with water hygiene issues.³ This is due to the alteration of the nutrient composition of their habitat to one which is optimal for growth (or blooms). Cyanobacterial blooms may simply cause foul tastes and odours,⁴ but they can also lead to the production of toxic secondary metabolites poisonous to humans and animals upon ingestion.⁵ NZ has yet to suffer a human fatality, but the deaths of several dogs in Wellington was attributed to homoanatoxin-a 1 (Chart 1) from a Phormidium species.⁶

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  • Potent toxins in Arctic environments – Presence of saxitoxins and an unusual microcystin variant in Arctic freshwater ecosystems

    Kleinteich, Julia; Wood, Susanna A.; Puddick, Jonathan; Schleheck, David; Küpper, Frithjof C.; Dietrich, Daniel R. (2013)

    Journal article
    University of Waikato

    Cyanobacteria are the predominant phototrophs in freshwater ecosystems of the polar regions where they commonly form extensive benthic mats. Despite their major biological role in these ecosystems, little attention has been paid to their physiology and biochemistry. An important feature of cyanobacteria from the temperate and tropical regions is the production of a large variety of toxic secondary metabolites. In Antarctica, and more recently in the Arctic, the cyanobacterial toxins microcystin and nodularin (Antarctic only) have been detected in freshwater microbial mats. To date other cyanobacterial toxins have not been reported from these locations. Five Arctic cyanobacterial communities were screened for saxitoxin, another common cyanobacterial toxin, and microcystins using immunological, spectroscopic and molecular methods. Saxitoxin was detected for the first time in cyanobacteria from the Arctic. In addition, an unusual microcystin variant was identified using liquid chromatography–mass spectrometry. Gene expression analyses confirmed the analytical findings, whereby parts of the sxt and mcy operon involved in saxitoxin and microcystin synthesis, were detected and sequenced in one and five of the Arctic cyanobacterial samples, respectively. The detection of these compounds in the cryosphere improves the understanding of the biogeography and distribution of toxic cyanobacteria globally. The sequences of sxt and mcy genes provided from this habitat for the first time may help to clarify the evolutionary origin of toxin production in cyanobacteria.

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  • A comparative proteomics study of skeletal muscle mitochondria from myostatin null mice

    Puddick, Jonathan; Martinus, Ryan Dennis (2011)

    Journal article
    University of Waikato

    Myostatin is a secreted protein which is known to be a negative regulator of skeletal muscle growth. Numerous studies have demonstrated that down-regulating the expression of myostatin results in an increase in muscle mass. This increase in skeletal muscle is accompanied by a marked change in the muscle fibre composition from one reliant on mitochondrial oxidative metabolism to one more focused on glycolytic metabolism. A comparative proteomics study was undertaken to investigate the effect of this altered metabolism has on the mitochondria from the gastrocnemius muscle of myostatin null mice, compared with those from wild type mice. The majority of the proteins identified showed no significant modulation between the two phenotypes, but gives an interesting insight into observations made in previous studies. Several proteins were shown to be modulated; however, only one of these was able to be identified. This protein which had sequence similarity to aldehyde reductase, was up-regulated in myostatin null mitochondria. The significance of this observation remains to be established. Interestingly, this protein has been implicated in detoxification of harmful products of lipid peroxidation.

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  • Hantupeptins B and C, cytotoxic cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula

    Tripathi, Ashootosh; Puddick, Jonathan; Prinsep, Michèle R.; Lee, Peter Peng Foo; Tan, Lik Tong (2010-02)

    Journal article
    University of Waikato

    Hantupeptins B (2) and C (3) were isolated, along with the previously reported hantupeptin A (1), from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu Besar, Singapore. Their structures were elucidated by interpretation of extensive 1D and 2D NMR spectroscopic data. Compounds 2 and 3 are cyclic depsipeptides consisting of five α-amino/hydroxy acid residues, including phenyllactic acid, proline, N-methyl-valine, valine, N-methyl-isoleucine, and a β-hydroxy acid unit with different degrees of unsaturation at the terminal end of each molecule. The absolute configurations of the common amino acids and phenyllactic acid were determined by the advanced Marfey’s and chiral HPLC analyses, respectively. The complete stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid moiety in hantupeptin A was elucidated by a combination of homonuclear J-resolved 2D NMR experiments and by Mosher’s method. Hantupeptins B and C showed moderate in vitro cytotoxicity when tested against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines.

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  • Isolation and structure determination of two new hydrophobic microcystins from Microcystis sp. (CAWBG11)

    Puddick, Jonathan; Prinsep, Michèle R.; Wood, Susanna A.; Cary, S. Craig; Hamilton, David P.; Wilkins, Alistair L. (2013)

    Journal article
    University of Waikato

    Two new hydrophobic microcystins, microcystin-FA (1) and microcystin-WA (2), were isolated from the cyanobacterium Microcystis sp. (CAWBG11). The structures were deduced using one- and two-dimensional nuclear magnetic resonance spectroscopy and tandem mass spectrometry. The absolute stereochemistry of the amino acid residues in 1 and 2 was determined using the Advanced Marfey's method.

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  • Further characterization of glycine-containing microcystins from the McMurdo Dry Valleys of Antarctica

    Puddick, Jonathan; Prinsep, Michèle R.; Wood, Susanna A.; Cary, S. Craig; Hamilton, David P.; Holland, Patrick T. (2015-02-10)

    Journal article
    University of Waikato

    Microcystins are hepatotoxic cyclic peptides produced by several cyanobacterial genera worldwide. In 2008, our research group identified eight new glycine-containing microcystin congeners in two hydro-terrestrial mat samples from the McMurdo Dry Valleys of Eastern Antarctica. During the present study, high-resolution mass spectrometry, amino acid analysis and micro-scale thiol derivatization were used to further elucidate their structures. The Antarctic microcystin congeners contained the rare substitution of the position-1 D-alanine for glycine, as well as the acetyl desmethyl modification of the position-5 Adda moiety (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid). Amino acid analysis was used to determine the stereochemistry of several of the amino acids and conclusively demonstrated the presence of glycine in the microcystins. A recently developed thiol derivatization technique showed that each microcystin contained dehydrobutyrine in position-7 instead of the commonly observed N-methyl dehydroalanine.

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  • The effect of cyanobacterial biomass enrichment by centrifugation and GF/C filtration on subsequent microcystin measurement

    Rogers, Shelley; Puddick, Jonathan; Wood, Susanna A.; Dietrich, Daniel R.; Hamilton, David P.; Prinsep, Michèle R. (2015-03)

    Journal article
    University of Waikato

    Microcystins are cyclic peptides produced by multiple cyanobacterial genera. After accumulation in the liver of animals they inhibit eukaryotic serine/threonine protein phosphatases, causing liver disease or death. Accurate detection/quantification of microcystins is essential to ensure safe water resources and to enable research on this toxin. Previous methodological comparisons have focused on detection and extraction techniques, but have not investigated the commonly used biomass enrichment steps. These enrichment steps could modulate toxin production as recent studies have demonstrated that high cyanobacterial cell densities cause increased microcystin levels. In this study, three microcystin-producing strains were processed using no cell enrichment steps (by direct freezing at three temperatures) and with biomass enrichment (by centrifugation or GF/C filtration). After extraction, microcystins were analyzed using liquid chromatography-tandem mass spectrometry. All processing methods tested, except GF/C filtration, resulted in comparable microcystin quotas for all strains. The low yields observed for the filtration samples were caused by adsorption of arginine-containing microcystins to the GF/C filters. Whilst biomass enrichment did not affect microcystin metabolism over the time-frame of normal sample processing, problems associated with GF/C filtration were identified. The most widely applicable processing method was direct freezing of samples as it could be utilized in both field and laboratory environments.

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  • Fine-scale cryogenic sampling of planktonic microbial communities: Application to toxic cyanobacterial blooms

    Puddick, Jonathan; Wood, Susanna A.; Hawes, Ian; Hamilton, David P. (2016)

    Journal article
    University of Waikato

    A lack of fine-scale methods for sampling planktonic microbial populations hinders advancement in understanding the responses of these communities to environmental conditions. Current methods provide resolution at scales of centimeters to meters, but not at the millimeter-scale required to understand highly stratified communities. To address this we developed two cryogenic sampling tools to collect spatially-precise samples from aquatic environments while simultaneously preserving the microbial communities. The application of these samplers was examined over a 5.5 h period using a cyanobacterial scum (Microcystis) formed in experimental mesocosms. A cryogenic “surface snatcher” collected a discrete layer (ca. 1 mm) of surface water. Compared to conventional surface sampling methods, the surface snatcher samples contained up to 22-times more microcystin, indicating that less underlying water was incorporated into the sample. A cryogenic “cold finger” sampler was used to collect vertical profiles of the upper 40 mm of the water column. This profiler provided new insights into the fine-scale structure of Microcystis scums, demonstrating that more microcystin-producing Microcystis was contained in the surface 5 mm than the 35 mm below. The results also showed that upregulation of microcystin production was highly localized in the top 2.5 mm of the Microcystis scum. Our results demonstrate that extreme changes in cyanobacterial communities can occur over small distances, and indicate that sampling resolution is of great importance for improving knowledge on cyanobacterial blooms and toxin production. While this study focused on microcystin-producing Microcystis, the cryogenic sampling tools described here could be applied to any planktonic microbial community.

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  • Widespread distribution and identification of eight novel microcystins in Antarctic cyanobacterial mats

    Wood, Susanna A.; Mountfort, Douglas O.; Selwood, Andrew I.; Holland, Patrick T.; Puddick, Jonathan; Cary, S. Craig (2008)

    Journal article
    University of Waikato

    The microcystin content and cyanobacterial community structure of Antarctic microbial mat samples collected from 40 ponds, lakes and hydro-terrestrial environments were investigated. Samples were collected from Bratina Island and four of the Dry Valleys; Wrights, Victoria, Miers and Marshall. Enzyme-linked immunosorbent assays (ELISA), liquid chromatography mass spectrometry (LC-MS), and protein phosphatase inhibition assays (PP-2A) resulted in the identification of low levels (1 - 16 mg/kg dry weight) of microcystins in all samples. A plot of indicative potencies of microcystins (ratio PP-2A:ELISA) versus total microcystins (ELISA) showed a general decrease in potency as total microcystin levels increased and a clustering of values from discrete geographic locations. LC-MS/MS analysis on selected samples identified eight novel microcystin congeners. The low energy collisional activation spectra were consistent with variants of [D-Asp3] MC-RR and [D-Asp3] MC-LR containing glycine [Gly1] rather than alanine and combinations of homoarginine [hAr2] or acetyldemethyl ADDA [ADMAdda5] substitutions. Nostoc sp. was identified as a microcystin producer using PCR amplification of a region of the 16S rRNA gene and the aminotransferase (AMT) domain of the mcyE gene. Automated ribosomal intergenic spacer analysis (ARISA) was undertaken to enable a comparison of cyanobacterial mat community structure from distant geographical locations. Two-dimensional multidimensional scaling ordination analysis of the ARISA data showed that in general samples from the same geographic location tended to clustered together. ARISA also enabled the putative identification of the microcystin producing Nostoc sp. from multiple samples.

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  • Structural characterization of new microcystins containing tryptophan and oxidized tryptophan residues

    Puddick, Jonathan; Prinsep, Michèle R.; Wood, Susanna A.; Miles, Christopher O.; Rise, Frode; Cary, S. Craig; Hamilton, David P.; Wilkins, Alistair L. (2013)

    Journal article
    University of Waikato

    Microcystins are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Eight of the (more than) 90 microcystin variants presently characterized, contain the amino acid tryptophan. The well-researched oxidation products of tryptophan; kynurenine, oxindolylalanine, and N-formylkynurenine, have been previously identified in intact polypeptides but microcystin congeners containing oxidized tryptophan moieties have not been reported. Liquid chromatography-tandem mass spectrometric analysis of an extract of Microcystis CAWBG11 led to the tentative identification of two new tryptophan-containing microcystins (MC‑WAba and MC-WL), as well as eight other microcystin analogs containing kynurenine, oxindolylalanine and N‑formylkynurenine (Nfk). Investigation of one of these congeners (MC‑NfkA) by nuclear magnetic resonance spectroscopy was used to verify the presence of Nfk in the microcystin. Liquid chromatography-mass spectrometry analysis of a tryptophan oxidation experiment demonstrated that tryptophan-containing microcystins could be converted into oxidized tryptophan analogs and that low levels of oxidized tryptophan congeners were present intracellularly in CAWBG11. MC-NfkR and MC-LNfk were detected in standards of MC-WR and MC-LW, indicating that care during storage of tryptophan-containing microcystins is required.

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  • Enhanced sample preparation for quantitation of microcystins by matrix-assisted laser desorption/ionisation–time of flight mass spectrometry

    Puddick, Jonathan; Prinsep, Michèle R.; Wood, Susanna A.; Cary, S. Craig; Hamilton, David P. (2011)

    Journal article
    University of Waikato

    Introduction: Microcystins (MCs) are a group of cyanotoxins which pose a serious health threat when present in aquatic systems. Quantitative analysis of MCs by matrix-assisted laser desorption/ionisation–time of flight (MALDI–TOF) mass spectrometry has potential for the processing of large numbers of samples quickly and economically. The existing method uses an expensive internal standard and protocols that are incompatible with automated sample preparation and data acquisition. Objective: To produce a MALDI–TOF sample preparation technique for the quantitation of MCs that not only maintains reproducibility and sensitivity, but is also compatible with an automated work-flow. Methodology: Seven different MALDI–TOF sample preparations were assessed for signal reproducibility (coefficient of variation) and sensitivity (method detection limit) using a cost-effective internal standard (angiotensin I). The best preparation was then assessed for its quantitative performance using three different MC congeners ([Dha7] MC-LR, MC-RR and MC-YR). Results: The sensitivity of six of the preparations was acceptable, as was the reproducibility for two thin-layer preparations performed on a polished steel target. Both thin-layer preparations could be used with a MALDI-TOF mass spectrometer that automatically acquires data, and one could be used in an automated sample preparation work-flow. Further investigation using the thin-layer spot preparation demonstrated that linear quantification of three different MC congeners was possible. Conclusion: The study demonstrates that with different sample preparation methods and modern instrumentation, large numbers of samples can be analysed rapidly for MCs at low cost.

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  • Laser desorption ionisation–time of flight mass spectrometry of the tolyporphins, bioactive metabolites from the cyanobacterium Tolypothrix nodosa

    Prinsep, Michèle R.; Puddick, Jonathan (2011)

    Journal article
    University of Waikato

    Introduction – The tolyporphins are metabolites isolated from the cyanobacterium Tolypothrix nodosa, comprising a porphyrin-like macrocycle with C-glycoside, hydroxide or acetate substituents. Previous studies of porphyrins by MALDI/LDI-TOF MS indicate that strong radical cations and anions are usually observed in the parent spectra with little fragmentation of the macrocycle. The spectra of the tolyporphins were obtained and trends in the series utilised to partially characterise two new analogues. Objective – To examine tolyporphins by LDI-TOF MS and utilise trends observed to partially characterise two new analogues. Methodology – The tolyporphins were analysed by LDI-TOF MS in positive and negative ion mode and by a post source decay method (LIFT) in positive ion mode. Tolyporphin A was also analysed by MALDI-TOF MS for comparison. Results were analysed and used to obtain structural information on two new analogues. Results and Discussion – The resulting spectra generally contained intense radical cations or anions, with little fragmentation of the macrocyclic core or the C-glycosides observed. These results are consistent with previous studies of porphyrins. Major fragment ions observed in LIFT spectra yielded key structural information. An inseparable mixture of two tolyporphins was also examined. Analysis of the LIFT spectrum of the parent ion resulted in the postulation of structures of these two new analogues. Conclusions – Tolyporphins yield LDI-TOF mass spectra somewhat analogous to those of porphyrins; furthermore, the substituents fragment in a characteristic manner permitting partial characterisation of the new analogues tolyporphins L and M by comparison of their LDI-TOF mass spectra with those of the known analogues.

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  • Lagunamides A and B: Cytotoxic and antimalarial Cyclodepsipeptides from the Marine cyanobacterium Lyngbya majuscule

    Tripathi, Ashootosh; Puddick, Jonathan; Prinsep, Michèle R.; Rottmann, Matthias; Tan, Lik Tong (2010)

    Journal article
    University of Waikato

    Lagunamides A (1) and B (2) are new cyclic depsipeptides isolated from the marine cyanobacterium Lyngbya majuscula obtained from Pulau Hantu Besar, Singapore. The planar structural characterization of these molecules was achieved by extensive spectroscopic analysis, including 2D NMR experiments. In addition to Marfey’s method and 3JH−H coupling constant values, a modified method based on Mosher’s reagents and analysis using LC-MS was deployed for the determination of the absolute configuration. Lagunamides A and B displayed significant antimalarial properties, with IC₅₀ values of 0.19 and 0.91 μM, respectively, when tested against Plasmodium falciparum. Lagunamides A and B also possessed potent cytotoxic activity against P388 murine leukemia cell lines, with IC₅₀ values of 6.4 and 20.5 nM, respectively. Furthermore, these cyanobacterial compounds exhibited moderate antiswarming activities when tested against Pseudomonas aeruginosa PA01.

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  • Hantupeptin A, a cytotoxic cyclic depsipeptide from a Singapore collection of Lyngbya majuscula

    Tripathi, Ashootosh; Puddick, Jonathan; Prinsep, Michèle R.; Lee, Peter Peng Foo; Tan, Lik Tong (2009)

    Journal article
    University of Waikato

    Chemical investigation of the marine cyanobacterium Lyngbya majuscula from Pulau Hantu Besar, Singapore, has led to the isolation of a cyclodepsipeptide, hantupeptin A (1). The planar structure of 1 was assigned on the basis of extensive 1D and 2D NMR spectroscopic experiments. The absolute configuration of the amino and hydroxyl acid residues in the molecule was determined by application of the advanced Marfey method, chiral HPLC analysis, and Mosher’s method. Hantupeptin A showed cytotoxicity to MOLT-4 leukemia cells and MCF-7 breast cancer cells with IC50 values of 32 and 4.0 µM, respectively.

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