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

authors: Puddick, Jonathan

subjects: Microcystins

abstract: 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.

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

year: 2013

title: Coastal Storm Activity along the Eastern North Island of New Zealand - East Cape to Wellington

authors: Dunn, Amber Susan

subjects: coastal storms

abstract: Coastal storm activity for the eastern North Island, between East Cape and Wellington, has been quantified from a meteorological perspective through the use of cyclone tracks and extreme winds and from an oceanographic perspective by using hindcast wave information. It has culminated in the production of a high quality, digital coastal storm database for the eastern North Island. Together, this information provides a new understanding of coastal storm behaviour for the eastern North Island. A regional database of historical coastal storms along the eastern North Island between 1930 and 2005 (75 years) is now available in digital format. Coastal storms were identified as bouts of strong winds (greater than or equal to 10.5 m/s) from long-term local wind records from 1962 to 2005, and prior to this period, coastal storms were qualitatively recognised as any event leading to coastal shipping disruptions/delays, large wave conditions along the coast, episodes of coastal erosion and strong onshore wind periods. This digital database consists of five informative components that include storm meteorology, storm oceanography, impacts and damages, storm photo’s and images, and data sources. It has identified a set of five storm types for the eastern North Island consisting of Trough/Ridges, East Coast Lows, Subtropical Lows, Tasman Sea Lows, and Cyclone-Anticyclone pair. The two dominant types are Trough/Ridge and East Coast Low, with the Trough pattern involving weather systems primarily from the southern ocean, whilst East Coast Lows involve large cyclones off the coast that can be distantly generated (from the Tasman Sea or subtropics) or locally generated around NZ from southern ocean troughs. The most intense coastal storms off the eastern North Island are East Coast Lows involving cyclones from the subtropics. These storm events reveal blocking-type anticyclones east of the Chatham Islands play a vital role in coastal storm activity by steering cyclones southward towards NZ and then blocking any eastward movements so that cyclones become slow-moving off the east coast. These factors increase the intensity of pressure gradients directly over eastern NZ. The Gisborne region, for the 1962-1991 period (30 years), had an annual average of three coastal storms and displays peak activity in September. These storms are overwhelming from the south and southeast. A longer dataset of local winds at Wellington, spanning 1962-2005 (44 years), produced an annual average frequency of 9 coastal storms per year. The monthly distribution revealed peak storm activity in June and heightened activity between May and August. Both short-lived, high intensity storms (winds greater than or equal to 14.5 m/s for at least 12 hours) and long-lived, lower intensity storms (winds greater than or equal to 10.5m/s for at least 24 hours) were identified for the Wellington region. Approximately 70% of these coastal storms persisted for up to two days duration and are predominately from the south and southwest. Furthermore, the more exposed nature and steep terrain surrounding Wellington means a greater likelihood of higher intensity coastal storms compared to the Gisborne region. Strong cyclonic systems in the southwest Pacific cluster in the central Tasman Sea and east of the Chatham Islands in all seasons and are most frequent in winter. It is during winter that a clear frequency maximum is spotted over North Cape and appears to be related to the presence of slow-moving cyclones rather than high counts of discrete systems. Strong cyclones tend to form in the western Tasman Sea, in the subtropics near 22-23S, and near North Cape. This local formation off North Cape could be related to the Tasman front and North Cape eddy which create warm sea surface temperature anomalies. The complete life cycle of all strong cyclones shows formation, intensification and maturity in the western-central Tasman Sea, and therefore, a large proportion of these cyclones approaching NZ are weakening systems. However, local generation and intensification near North Cape and the Chatham Islands ensures strong cyclones continue to influence eastern NZ, and further indicates weakened Tasman Sea cyclones can drive coastal storm events through interactions with ridges and high pressure systems. Strong cyclones are most frequent around NZ in August when an average of 4-5 systems per month occurs. Extreme onshore winds off the eastern coast of NZ consist principally of winds from the southwest and south with a single high latitude frequency maximum near the dateline. These winds are generated from southern ocean cyclonic activity and their northward-extending troughs that pass over NZ, and their spread onto eastern NZ means they likely represent intense coastal storm events. Southeast, east and northeast winds rarely reach up to and beyond 20 m.s-1 over the seas to the east of NZ and generally cluster north of 40-45S indicating both subtropical and higher midlatitude source mechanisms. Extreme southeast winds are generated by the eastern flanks of large anticyclones that occupy the western Tasman Sea or large anticyclones south of the Chatham Islands. The principal frequency maximum of east and northeast winds is remote from NZ appearing near 165°W, and represent distant generation areas for large swell events (rather than coastal storm activity). These winds that occur over northern NZ are associated with a Tasman Sea or subtropical cyclone off North Cape in combination with a large anticyclone or ridge over/or east of the South Island. In contrast, the distant core for eastward of NZ are generated off the backs of large anticyclones with a trough or cyclone on its northern flank. The deep-water wave climate off the eastern North Island is dominated by waves from the south. Between 9 and 13 large wave events occur each year between East Cape and Wellington and are most likely in the months of May, June and July. In contrast, large storm waves from the southeast, east and northeast have annual average frequencies of 1-3 events. The Gisborne coast was found to be the most exposed with large deep-water waves (greater than or equal to 3m) coming from the northeast through to the southwest. However, waves from the south and southwest are the largest and most persistent. The meteorology creating these waves are southern ocean troughs whilst the less frequent waves from the easterly quarter involve low pressure systems east or northeast of NZ. The different proxies for studying coastal storms all have shortfalls and arrive at different levels of coastal storm activity. It is suggested here that an optimal mix of these proxies can be used to identify damaging coastal storms along the eastern North Island.

text: Coastal Storm Activity along the Eastern North Island of New Zealand - East Cape to Wellington Coastal storm activity for the eastern North Island, between East Cape and Wellington, has been quantified from a meteorological perspective through the use of cyclone tracks and extreme winds and from an oceanographic perspective by using hindcast wave information. It has culminated in the production of a high quality, digital coastal storm database for the eastern North Island. Together, this information provides a new understanding of coastal storm behaviour for the eastern North Island. A regional database of historical coastal storms along the eastern North Island between 1930 and 2005 (75 years) is now available in digital format. Coastal storms were identified as bouts of strong winds (greater than or equal to 10.5 m/s) from long-term local wind records from 1962 to 2005, and prior to this period, coastal storms were qualitatively recognised as any event leading to coastal shipping disruptions/delays, large wave conditions along the coast, episodes of coastal erosion and strong onshore wind periods. This digital database consists of five informative components that include storm meteorology, storm oceanography, impacts and damages, storm photo’s and images, and data sources. It has identified a set of five storm types for the eastern North Island consisting of Trough/Ridges, East Coast Lows, Subtropical Lows, Tasman Sea Lows, and Cyclone-Anticyclone pair. The two dominant types are Trough/Ridge and East Coast Low, with the Trough pattern involving weather systems primarily from the southern ocean, whilst East Coast Lows involve large cyclones off the coast that can be distantly generated (from the Tasman Sea or subtropics) or locally generated around NZ from southern ocean troughs. The most intense coastal storms off the eastern North Island are East Coast Lows involving cyclones from the subtropics. These storm events reveal blocking-type anticyclones east of the Chatham Islands play a vital role in coastal storm activity by steering cyclones southward towards NZ and then blocking any eastward movements so that cyclones become slow-moving off the east coast. These factors increase the intensity of pressure gradients directly over eastern NZ. The Gisborne region, for the 1962-1991 period (30 years), had an annual average of three coastal storms and displays peak activity in September. These storms are overwhelming from the south and southeast. A longer dataset of local winds at Wellington, spanning 1962-2005 (44 years), produced an annual average frequency of 9 coastal storms per year. The monthly distribution revealed peak storm activity in June and heightened activity between May and August. Both short-lived, high intensity storms (winds greater than or equal to 14.5 m/s for at least 12 hours) and long-lived, lower intensity storms (winds greater than or equal to 10.5m/s for at least 24 hours) were identified for the Wellington region. Approximately 70% of these coastal storms persisted for up to two days duration and are predominately from the south and southwest. Furthermore, the more exposed nature and steep terrain surrounding Wellington means a greater likelihood of higher intensity coastal storms compared to the Gisborne region. Strong cyclonic systems in the southwest Pacific cluster in the central Tasman Sea and east of the Chatham Islands in all seasons and are most frequent in winter. It is during winter that a clear frequency maximum is spotted over North Cape and appears to be related to the presence of slow-moving cyclones rather than high counts of discrete systems. Strong cyclones tend to form in the western Tasman Sea, in the subtropics near 22-23S, and near North Cape. This local formation off North Cape could be related to the Tasman front and North Cape eddy which create warm sea surface temperature anomalies. The complete life cycle of all strong cyclones shows formation, intensification and maturity in the western-central Tasman Sea, and therefore, a large proportion of these cyclones approaching NZ are weakening systems. However, local generation and intensification near North Cape and the Chatham Islands ensures strong cyclones continue to influence eastern NZ, and further indicates weakened Tasman Sea cyclones can drive coastal storm events through interactions with ridges and high pressure systems. Strong cyclones are most frequent around NZ in August when an average of 4-5 systems per month occurs. Extreme onshore winds off the eastern coast of NZ consist principally of winds from the southwest and south with a single high latitude frequency maximum near the dateline. These winds are generated from southern ocean cyclonic activity and their northward-extending troughs that pass over NZ, and their spread onto eastern NZ means they likely represent intense coastal storm events. Southeast, east and northeast winds rarely reach up to and beyond 20 m.s-1 over the seas to the east of NZ and generally cluster north of 40-45S indicating both subtropical and higher midlatitude source mechanisms. Extreme southeast winds are generated by the eastern flanks of large anticyclones that occupy the western Tasman Sea or large anticyclones south of the Chatham Islands. The principal frequency maximum of east and northeast winds is remote from NZ appearing near 165°W, and represent distant generation areas for large swell events (rather than coastal storm activity). These winds that occur over northern NZ are associated with a Tasman Sea or subtropical cyclone off North Cape in combination with a large anticyclone or ridge over/or east of the South Island. In contrast, the distant core for eastward of NZ are generated off the backs of large anticyclones with a trough or cyclone on its northern flank. The deep-water wave climate off the eastern North Island is dominated by waves from the south. Between 9 and 13 large wave events occur each year between East Cape and Wellington and are most likely in the months of May, June and July. In contrast, large storm waves from the southeast, east and northeast have annual average frequencies of 1-3 events. The Gisborne coast was found to be the most exposed with large deep-water waves (greater than or equal to 3m) coming from the northeast through to the southwest. However, waves from the south and southwest are the largest and most persistent. The meteorology creating these waves are southern ocean troughs whilst the less frequent waves from the easterly quarter involve low pressure systems east or northeast of NZ. The different proxies for studying coastal storms all have shortfalls and arrive at different levels of coastal storm activity. It is suggested here that an optimal mix of these proxies can be used to identify damaging coastal storms along the eastern North Island.

year: 2010

title: Te tātari i te kaupapa

authors: Ka'ai, Tānia M.

abstract: The primary objective of this thesis focuses on the linking of the National Qualifications Framework and standards-based assessment as an emancipatory mechanism for Māori in Kaupapa Māori schooling and the wider Māori community in New Zealand.

Argued from an eclectic theoretical approach incorporating Kaupapa Māori theory, it espouses a position which rejects structural inequalities that perpetuate the subordinate positioning of Māori people through the development of culturally responsive assessment theories and practices in written language for teachers and children in Kura Kaupapa Māori which purport a culturally responsive pedagogy.

The thesis also links curriculum, assessment and pedagogy using Kaupapa Māori theory which provides insights into Māori views of emancipation in education.

text: Te tātari i te kaupapa The primary objective of this thesis focuses on the linking of the National Qualifications Framework and standards-based assessment as an emancipatory mechanism for Māori in Kaupapa Māori schooling and the wider Māori community in New Zealand.

Argued from an eclectic theoretical approach incorporating Kaupapa Māori theory, it espouses a position which rejects structural inequalities that perpetuate the subordinate positioning of Māori people through the development of culturally responsive assessment theories and practices in written language for teachers and children in Kura Kaupapa Māori which purport a culturally responsive pedagogy.

The thesis also links curriculum, assessment and pedagogy using Kaupapa Māori theory which provides insights into Māori views of emancipation in education.

year: 1995