title: Toward catalytic antibodies capable of ergopeptine hydrolysis

authors: Dines, Mark

abstract: Animals grazing endophyte infected pasture are exposed to the toxic effects of ergopeptine compounds which are produced by the grass-endophyte interaction. Grasses containing endophytic fungi have a competitive advantage and comprise over 60% of pastureland. Catalytic antibody vaccines would permit the use of endophyte-infected grasses without the risk of animal toxicoses. The production of catalytic antibodies capable of hydrolysing ergopeptine toxins was investigated. The literature pertaining to catalytic antibodies is reviewed with particular focus on hydrolytic reactions. Model systems were used to investigate the potential of this technique.

Model phosphorus-ester and -amide compounds were synthesised and characterised. Problems were experienced forming the RPO(OCH₂CH₃)X where X is NH(C₆H₄NO₂), and R is a protected benzyl system, successful generation of model compounds where X is NHCH₂(C₆H₅) and O(C₆H₄NO₂) was achieved. The compound HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) was synthesised as a transition-state emulator for the generation of polyclonal antibodies capable of hydrolysing (C₆H₅)CH₂COO(C₆H₄NO₂) and (C₆H₅)CH₂CONH(C₆H₄NO₂). The transition-state emulator was conjugated to Bovine Serum Albumin, Thyroglobulin and Keyhole Limpet Haemocyanin. Sheep, mice and a rabbit were immunised with the resulting conjugate material.

The immune rabbit serum was found to rapidly hydrolyse ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) relative to the non-immune serum, however, purification of the immune rabbit antibodies indicated that the hydrolysis of ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) was not due to antibody catalysis. The non-purified sera from the immune sheep and mice was found to hydrolyse ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) at a similar rate to the non-immune sera and no immune sera was found to hydrolyse amide ((C₆H₅)CH₂CONH(C₆H₄NO₂)). Investigation of sera from immunised animals by ELISA indicated production of polyclonal antibodies capable of binding the HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) material conjugated to protein. This protein was different to that used as immunogen. However, the non-conjugated HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) material did not competitively inhibit polyclonal antibody binding to the protein conjugates, suggesting that the polyclonal antibodies generated required the presence of a conjugated protein environment for binding.
 
An investigation of the ergot alkaloids produced by one grass-endophyte association (Acnatherum inebrians) known to contain high levels of ergot alkaloid was conducted. Extraction methodology and reverse phase HPLC separation systems were developed for these compounds. Use of these methods enabled the purification of iso-lysergic acid amide and ergonovinine from Acnatherum inebrians. These compounds were characterised using NMR.

This thesis details the generation of a model transition-state compound and subsequent immunisation of this compound into animals. Antibodies from the immune animals were not found to catalyse the hydrolysis of amide or ester bonds. This work and the work of others suggests that a better understanding of the emulation of the amide transition-state is required before detoxification of ergopeptines by amide hydrolysising antibodies will be achieved.

text: Toward catalytic antibodies capable of ergopeptine hydrolysis Animals grazing endophyte infected pasture are exposed to the toxic effects of ergopeptine compounds which are produced by the grass-endophyte interaction. Grasses containing endophytic fungi have a competitive advantage and comprise over 60% of pastureland. Catalytic antibody vaccines would permit the use of endophyte-infected grasses without the risk of animal toxicoses. The production of catalytic antibodies capable of hydrolysing ergopeptine toxins was investigated. The literature pertaining to catalytic antibodies is reviewed with particular focus on hydrolytic reactions. Model systems were used to investigate the potential of this technique.

Model phosphorus-ester and -amide compounds were synthesised and characterised. Problems were experienced forming the RPO(OCH₂CH₃)X where X is NH(C₆H₄NO₂), and R is a protected benzyl system, successful generation of model compounds where X is NHCH₂(C₆H₅) and O(C₆H₄NO₂) was achieved. The compound HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) was synthesised as a transition-state emulator for the generation of polyclonal antibodies capable of hydrolysing (C₆H₅)CH₂COO(C₆H₄NO₂) and (C₆H₅)CH₂CONH(C₆H₄NO₂). The transition-state emulator was conjugated to Bovine Serum Albumin, Thyroglobulin and Keyhole Limpet Haemocyanin. Sheep, mice and a rabbit were immunised with the resulting conjugate material.

The immune rabbit serum was found to rapidly hydrolyse ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) relative to the non-immune serum, however, purification of the immune rabbit antibodies indicated that the hydrolysis of ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) was not due to antibody catalysis. The non-purified sera from the immune sheep and mice was found to hydrolyse ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) at a similar rate to the non-immune sera and no immune sera was found to hydrolyse amide ((C₆H₅)CH₂CONH(C₆H₄NO₂)). Investigation of sera from immunised animals by ELISA indicated production of polyclonal antibodies capable of binding the HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) material conjugated to protein. This protein was different to that used as immunogen. However, the non-conjugated HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) material did not competitively inhibit polyclonal antibody binding to the protein conjugates, suggesting that the polyclonal antibodies generated required the presence of a conjugated protein environment for binding.
 
An investigation of the ergot alkaloids produced by one grass-endophyte association (Acnatherum inebrians) known to contain high levels of ergot alkaloid was conducted. Extraction methodology and reverse phase HPLC separation systems were developed for these compounds. Use of these methods enabled the purification of iso-lysergic acid amide and ergonovinine from Acnatherum inebrians. These compounds were characterised using NMR.

This thesis details the generation of a model transition-state compound and subsequent immunisation of this compound into animals. Antibodies from the immune animals were not found to catalyse the hydrolysis of amide or ester bonds. This work and the work of others suggests that a better understanding of the emulation of the amide transition-state is required before detoxification of ergopeptines by amide hydrolysising antibodies will be achieved.

year: 1998

title: Keeping pace with technology: drones, disturbance and policy deficiency

authors: Wallace, Philippa Jane

subjects: UAV

abstract: This paper analyses regulatory responses to rapid intensiﬁcation of the use of drones/remotely piloted aircraft (RPA) in the context of wildlife protection. Beneﬁts and disadvantages of the technology to wildlife are examined, before three key limitations in policy and law are identiﬁed: failure to address wildlife disturbance in RPA regulation; reliance upon insufﬁciently comprehensive existing wildlife protection legislation to manage disturbance effects; and limited species-speciﬁc research on disturbance. A New Zealand case study further reveals an inconsistent regulatory approach struggling to keep pace with innovation, inadequate regulatory capture of environmental effects due to exemption as “aircraft”, and no recognition that speciﬁc geographical locations, such as coastal areas, distinguished by recreational pressures and high numbers of threatened species require special consideration. Recommendations include acknowledging the impact on wildlife in policy, gap analysis of legal arrangements for protection from disturbance (including airspace), and adoption of minimum approach distances to threatened species.

text: Keeping pace with technology: drones, disturbance and policy deficiency This paper analyses regulatory responses to rapid intensiﬁcation of the use of drones/remotely piloted aircraft (RPA) in the context of wildlife protection. Beneﬁts and disadvantages of the technology to wildlife are examined, before three key limitations in policy and law are identiﬁed: failure to address wildlife disturbance in RPA regulation; reliance upon insufﬁciently comprehensive existing wildlife protection legislation to manage disturbance effects; and limited species-speciﬁc research on disturbance. A New Zealand case study further reveals an inconsistent regulatory approach struggling to keep pace with innovation, inadequate regulatory capture of environmental effects due to exemption as “aircraft”, and no recognition that speciﬁc geographical locations, such as coastal areas, distinguished by recreational pressures and high numbers of threatened species require special consideration. Recommendations include acknowledging the impact on wildlife in policy, gap analysis of legal arrangements for protection from disturbance (including airspace), and adoption of minimum approach distances to threatened species.

year: 2017

title: Iwi sex ratios in the New Zealand population census: Why are women so dominant?

authors: Kukutai, Tahu

abstract: Recent census-based studies of iwi (tribal) population growth have revealed a high degree of volatility that cannot be explained by demographic factors alone. Although focused on a small number of iwi, these studies have shown that changing patterns of identification are an important driver of iwi population growth, and that the propensity to identify with an iwi appears to be much stronger among Māori women than men. Thus, the vast majority of iwi in the census have far more females than males, and female domination has increased over time. This paper describes the key features of female-favoured iwi sex ratios in the census and explores possible explanations. Focusing on sex ratios for the ten largest iwi, we find that female domination is highest in the 25–44 age group, and that this pattern is consistent over time. Further analysis shows that Māori women aged 25–34 years are more likely than their male counterparts to know detailed aspects of their pepeha (tribal identity), to explore whakapapa (genealogy) and to speak te reo Māori. Our results underscore the importance of Māori women as cultural connectors within their whānau, as well as in a broader iwi context.

text: Iwi sex ratios in the New Zealand population census: Why are women so dominant? Recent census-based studies of iwi (tribal) population growth have revealed a high degree of volatility that cannot be explained by demographic factors alone. Although focused on a small number of iwi, these studies have shown that changing patterns of identification are an important driver of iwi population growth, and that the propensity to identify with an iwi appears to be much stronger among Māori women than men. Thus, the vast majority of iwi in the census have far more females than males, and female domination has increased over time. This paper describes the key features of female-favoured iwi sex ratios in the census and explores possible explanations. Focusing on sex ratios for the ten largest iwi, we find that female domination is highest in the 25–44 age group, and that this pattern is consistent over time. Further analysis shows that Māori women aged 25–34 years are more likely than their male counterparts to know detailed aspects of their pepeha (tribal identity), to explore whakapapa (genealogy) and to speak te reo Māori. Our results underscore the importance of Māori women as cultural connectors within their whānau, as well as in a broader iwi context.

year: 2017