title: An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 degrees C.

authors: Cowan, Don A.

subjects: biology

abstract: An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5.

text: An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 degrees C. An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5.

year: 1987

title: Assessing mitochondrial DNA (CO1) barcodes for measuring diversity of Schizaphis spp. and abundance of Aploneura lentisci

authors: Podmore, Colleen

subjects: barcoding

abstract: We examined the diversity of mitochondrial DNA (CO1) sequences in the New Zealand native aphids with a particular focus on the genus Schizaphis (syn. Euschizaphis). Previously, the genus was thought to consist of two species, each host-specific to endemic New Zealand plants (Aciphylla and Dracophyllum). These unnamed native taxa are reasonably common but have either a narrow distribution (Aciphylla-host) or low local abundances (Dracophyllum-host). There is also some uncertainty over the number of Schizaphis species present on the various host plants. Specimens from both host plants were collected from ten sites in the North (n=3) and South (n=8) Islands. A total of 29 new COI sequences were obtained. A further 189 sequences were obtained from the Barcode of Life Datasystems (n=187) and GenBank (n=2). Thirteen of the 15 known native aphids were analysed by Maximum Likelihood and their taxonomic classification confirmed, the majority belonging to the Aphidinae, but also representatives in other sub-families. Maximum Likelihood analyses and pairwise genetic distances confirmed taxonomic groupings of the Aphididae sub-family Aphidinae and within the Aphidinae the monophyly of the sub-tribe Rhopalosiphina which contains two genera: Rhopalosiphum spp.and Schizaphis spp. Two distinct, well supported clades within Schizaphis were clearly delineated according to the host plant. However, we also found two distinct clusters within the Aciphylla-host South Island individuals and four within the Dracophyllum-host individuals (North & South Islands), suggesting six potentially cryptic species. Based on these data, we suggest that the CO1 gene region is effective for identifying aphids and could assist in the on-going discovery and protection of these taxa in New Zealand. New information regarding the diversity of New Zealand native Schizaphis aphids is using COI gene sequences to reveal additional diversity. International databases such as the Barcode of Life Datasystems and GenBank will be particularly helpful in examining global relationships within the New Zealand native aphid taxon.
Root Aphids (Aploneura lentisci) are an introduced taxon, which live exclusively on the roots of Poaceae and can cause detrimental effects to New Zealand pastures, reducing the forage available for farmed livestock. In order to assess abundances of A. lentisci in soil samples, specific primers were first developed to target and amplify the mitochondrial gene region cytochrome c oxidase subunit 1 (CO1) gene region. Three DNA extraction protocols, the PowerSoil® DNA Isolation Kit , Extract-N-Amp™Tissue PCR and a phenol chloroform DNA extraction protocol  were then assessed and real time quantitative polymerase chain reaction (qPCR) was used to measure the amount of amplicon produced, which was compared to a standard curve of known aphid numbers. The developed primers successfully amplified a 317 nucleotide fragment of the COI gene region and had limited cross reactivity with other aphid taxa tested. Of the three extraction methods, the Extract-N-Amp Tissue PCR Kit was the most effective. Adult root aphids were selected as representatives of infestations. DNA concentrations extracted using the Extract-N-Amp Kit were linear over the entire range of aphid numbers (R²=0.98). Further dilutions were carried out: 10-fold, 100-fold and 1000-fold to test linearity and when combined produced a standard curve with a regression of 0.93 over a cycle threshold range of 18-33 cycles. DNA extractions from mixtures of 60 aphids in soil produced amplifiable aphid DNA. We conclude that this method will successfully measure root aphid abundances from soil samples using mitochondrial CO1 gene sequences and qPCR.

text: Assessing mitochondrial DNA (CO1) barcodes for measuring diversity of Schizaphis spp. and abundance of Aploneura lentisci We examined the diversity of mitochondrial DNA (CO1) sequences in the New Zealand native aphids with a particular focus on the genus Schizaphis (syn. Euschizaphis). Previously, the genus was thought to consist of two species, each host-specific to endemic New Zealand plants (Aciphylla and Dracophyllum). These unnamed native taxa are reasonably common but have either a narrow distribution (Aciphylla-host) or low local abundances (Dracophyllum-host). There is also some uncertainty over the number of Schizaphis species present on the various host plants. Specimens from both host plants were collected from ten sites in the North (n=3) and South (n=8) Islands. A total of 29 new COI sequences were obtained. A further 189 sequences were obtained from the Barcode of Life Datasystems (n=187) and GenBank (n=2). Thirteen of the 15 known native aphids were analysed by Maximum Likelihood and their taxonomic classification confirmed, the majority belonging to the Aphidinae, but also representatives in other sub-families. Maximum Likelihood analyses and pairwise genetic distances confirmed taxonomic groupings of the Aphididae sub-family Aphidinae and within the Aphidinae the monophyly of the sub-tribe Rhopalosiphina which contains two genera: Rhopalosiphum spp.and Schizaphis spp. Two distinct, well supported clades within Schizaphis were clearly delineated according to the host plant. However, we also found two distinct clusters within the Aciphylla-host South Island individuals and four within the Dracophyllum-host individuals (North & South Islands), suggesting six potentially cryptic species. Based on these data, we suggest that the CO1 gene region is effective for identifying aphids and could assist in the on-going discovery and protection of these taxa in New Zealand. New information regarding the diversity of New Zealand native Schizaphis aphids is using COI gene sequences to reveal additional diversity. International databases such as the Barcode of Life Datasystems and GenBank will be particularly helpful in examining global relationships within the New Zealand native aphid taxon.
Root Aphids (Aploneura lentisci) are an introduced taxon, which live exclusively on the roots of Poaceae and can cause detrimental effects to New Zealand pastures, reducing the forage available for farmed livestock. In order to assess abundances of A. lentisci in soil samples, specific primers were first developed to target and amplify the mitochondrial gene region cytochrome c oxidase subunit 1 (CO1) gene region. Three DNA extraction protocols, the PowerSoil® DNA Isolation Kit , Extract-N-Amp™Tissue PCR and a phenol chloroform DNA extraction protocol  were then assessed and real time quantitative polymerase chain reaction (qPCR) was used to measure the amount of amplicon produced, which was compared to a standard curve of known aphid numbers. The developed primers successfully amplified a 317 nucleotide fragment of the COI gene region and had limited cross reactivity with other aphid taxa tested. Of the three extraction methods, the Extract-N-Amp Tissue PCR Kit was the most effective. Adult root aphids were selected as representatives of infestations. DNA concentrations extracted using the Extract-N-Amp Kit were linear over the entire range of aphid numbers (R²=0.98). Further dilutions were carried out: 10-fold, 100-fold and 1000-fold to test linearity and when combined produced a standard curve with a regression of 0.93 over a cycle threshold range of 18-33 cycles. DNA extractions from mixtures of 60 aphids in soil produced amplifiable aphid DNA. We conclude that this method will successfully measure root aphid abundances from soil samples using mitochondrial CO1 gene sequences and qPCR.

year: 2015

title: “Whānau Vibes”: Reclamation of self, space and community within group fitness gyms.

authors: Metekingi, Cydney

abstract: Group fitness gyms are becoming more popular in Aotearoa, New Zealand. This thesis is an investigation of the lived experiences of Māori members within a commercialised fitness space called BFT Hamilton East, located in Kirikiriroa, Hamilton. Drawing on Indigenous anthropology and autoethnography, and grounded in Kaupapa Māori theory, this thesis firstly explores how the gym becomes a site in which Māori members seek to reclaim the narrative around their own bodies. I explore the colonial depictions, the politics of the Indigenous body and how Māori-owned clothing brands contribute to this reclamation. I then examine how Māori gym members use the gym space with a sense of purpose, balancing the values of culture and commercialisation. And finally, I investigate the importance of relational well-being and the use of whanaungatanga and manaakitanga to create a sense of belonging among participants in their search for connection. In foregrounding Indigenous ways of knowing, being and moving in a commercial group fitness gym, this thesis aims to contribute to the anthropological conversations that advocate for the inclusion of Indigenous methodologies into Anthropology, centring Indigenous agency and advocating for the decolonisation of fitness spaces.

text: “Whānau Vibes”: Reclamation of self, space and community within group fitness gyms. Group fitness gyms are becoming more popular in Aotearoa, New Zealand. This thesis is an investigation of the lived experiences of Māori members within a commercialised fitness space called BFT Hamilton East, located in Kirikiriroa, Hamilton. Drawing on Indigenous anthropology and autoethnography, and grounded in Kaupapa Māori theory, this thesis firstly explores how the gym becomes a site in which Māori members seek to reclaim the narrative around their own bodies. I explore the colonial depictions, the politics of the Indigenous body and how Māori-owned clothing brands contribute to this reclamation. I then examine how Māori gym members use the gym space with a sense of purpose, balancing the values of culture and commercialisation. And finally, I investigate the importance of relational well-being and the use of whanaungatanga and manaakitanga to create a sense of belonging among participants in their search for connection. In foregrounding Indigenous ways of knowing, being and moving in a commercial group fitness gym, this thesis aims to contribute to the anthropological conversations that advocate for the inclusion of Indigenous methodologies into Anthropology, centring Indigenous agency and advocating for the decolonisation of fitness spaces.

year: 2025