title: Pterocellin A isolated from marine bryozoan Pterocella vesiculosa is cytotoxic to human HeLa cells via mitochondrial apoptotic processes

authors: Wang, Alice Tansi

abstract: Pterocellin A is a novel bioactive alkaloid isolated from the New Zealand marine bryozoan Pterocella vesiculosa. It exhibits potent antitumour activity towards the P388 (murine leukaemia) cell line in vitro and is selectively sensitive towards certain non-small cell lung, melanoma, and breast cancer cell lines, however, the biological mode of action of pterocellin A is unknown. Using the human cervical cancer cell line HeLa, we show that pterocellin A exhibited cytotoxicity against HeLa cells with an IC50 of 886 ng/mL. Time-course MTT and LDH assays were carried out and the results showed only a low level of cytosolic LDH was detected in the supernatant after all the cells have died from pterocellin A treatment at 2000 ng/mL. This indicated the cells maintained membrane integrity upon cell death which suggested apoptotic cell death. Additionally, morphological changes were observed under the microscope after 6 h of treatment. Cell shrinkage and nucleus condensation were observed, as well as apparent membrane blebbing, a key feature of apoptosis. The MTT data was also indicative of mitochondria impairment which could suggest that pterocellin A targets the mitochondria. This idea was supported by the observed changes in the morphology and location of the mitochondria after exposure to pterocellin A. Furthermore, the level of activated caspase-3 in HeLa cells increased after treatment with pterocellin A; activated caspase-3 can only be detected after a series of signalling events following the induction of apoptosis. These data support the notion that pterocellin A is an inducer of apoptosis in HeLa cells possibly via mitochondria related processes.

text: Pterocellin A isolated from marine bryozoan Pterocella vesiculosa is cytotoxic to human HeLa cells via mitochondrial apoptotic processes Pterocellin A is a novel bioactive alkaloid isolated from the New Zealand marine bryozoan Pterocella vesiculosa. It exhibits potent antitumour activity towards the P388 (murine leukaemia) cell line in vitro and is selectively sensitive towards certain non-small cell lung, melanoma, and breast cancer cell lines, however, the biological mode of action of pterocellin A is unknown. Using the human cervical cancer cell line HeLa, we show that pterocellin A exhibited cytotoxicity against HeLa cells with an IC50 of 886 ng/mL. Time-course MTT and LDH assays were carried out and the results showed only a low level of cytosolic LDH was detected in the supernatant after all the cells have died from pterocellin A treatment at 2000 ng/mL. This indicated the cells maintained membrane integrity upon cell death which suggested apoptotic cell death. Additionally, morphological changes were observed under the microscope after 6 h of treatment. Cell shrinkage and nucleus condensation were observed, as well as apparent membrane blebbing, a key feature of apoptosis. The MTT data was also indicative of mitochondria impairment which could suggest that pterocellin A targets the mitochondria. This idea was supported by the observed changes in the morphology and location of the mitochondria after exposure to pterocellin A. Furthermore, the level of activated caspase-3 in HeLa cells increased after treatment with pterocellin A; activated caspase-3 can only be detected after a series of signalling events following the induction of apoptosis. These data support the notion that pterocellin A is an inducer of apoptosis in HeLa cells possibly via mitochondria related processes.

year: 2016

title: Catabolism of naphthalene and phenanthrene by Burkholderia sp. strain RP007

authors: Laurie, Andrew David

abstract: Burkholderia sp. strain RP007 was isolated for its ability to utilise phenanthrene as a sole source of carbon and energy. This strain was identified as a Burkholderia sp. by biochemical tests, fatty acid analysis, and nucleotide sequencing of the 16S rRNA gene. The ability of Burkholderia sp. strain RP007 to mineralise [9-¹⁴C]phenanthrene and [1-¹⁴C]naphthalene was confirmed.

A gene cluster (phn) which represents a new group of upper pathway genes for polycyclic aromatic hydrocarbon (PAH) degradation was cloned from Burkholderia sp. strain RP007. Nucleotide sequencing of an 11.5 kb HindIII fragment cloned from RP007 revealed the presence of nine open reading frames (ORFѕ). Escherichia coli carrying the recombinant phn genes was able to transform naphthalene to salicylic acid. The phn genes are significantly different in sequence and gene order from the previously described nah, ndo, pah and dox genes for PAH degradation. The phn locus encodes iron-sulfur protein α and β subunits of a PAH initial dioxygenase, but not the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterised from naphthalene/phenanthrene pathways. Furthermore, the RP007 extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation, but is a member of the recently proposed Class III extradiol dioxygenases. Upstream of the phn catabolic genes are two putative regulatory genes phnR and phnS. phnR is divergently transcribed with respect to the other genes and is similar to the σ⁵⁴-dependent family of positive transcriptional regulators. phnS is a LysR-type transcriptional activator. Expression experiments suggest the phn operon is under regulatory control which may involve PhnR and PhnS.

Two loci encoding aromatic meta cleavage pathways were also cloned from Burkholderia sp. strain RP007. The nucleotide sequence of the catechol 2,3-dioxygenase genes, and adjacent open reading frames, suggest these are meta pathways of different classes which specify catabolism of different aromatic substrates. One of these may be the lower pathway for phenanthrene and naphthalene degradation.

The presence of catabolic genes homologous to the phnAc gene of Burkholderia sp. strain RP007, relative to the archetypal nahAc gene of Pseudomonas putida G7, was screened for in both culturable and nonculturable microbial communities. Of 77 environmental isolates cultured for their ability to degrade naphthalene or phenanthrene, 28 showed homology to nahAc, whilst none of the strains possessed catabolic genes similar to phnAc. However, using phnAc-specific primers, PCR products homologous to phnAc were amplified from DNA extracts of eight hydrocarbon-contaminated soils, demonstrating the widespread distribution of this genotype. Analysis of these phnAc PCR products by nucleotide sequencing of selected cloned products revealed very high (98.5-100%) homology to the phnAc gene of RP007, suggesting the phn genotype is highly conserved. PCR products were also amplified from soil DNA using nahAc-specific primers, and sequencing of these products revealed a diversity similar to that amongst previously described nah-like genes.

text: Catabolism of naphthalene and phenanthrene by Burkholderia sp. strain RP007 Burkholderia sp. strain RP007 was isolated for its ability to utilise phenanthrene as a sole source of carbon and energy. This strain was identified as a Burkholderia sp. by biochemical tests, fatty acid analysis, and nucleotide sequencing of the 16S rRNA gene. The ability of Burkholderia sp. strain RP007 to mineralise [9-¹⁴C]phenanthrene and [1-¹⁴C]naphthalene was confirmed.

A gene cluster (phn) which represents a new group of upper pathway genes for polycyclic aromatic hydrocarbon (PAH) degradation was cloned from Burkholderia sp. strain RP007. Nucleotide sequencing of an 11.5 kb HindIII fragment cloned from RP007 revealed the presence of nine open reading frames (ORFѕ). Escherichia coli carrying the recombinant phn genes was able to transform naphthalene to salicylic acid. The phn genes are significantly different in sequence and gene order from the previously described nah, ndo, pah and dox genes for PAH degradation. The phn locus encodes iron-sulfur protein α and β subunits of a PAH initial dioxygenase, but not the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterised from naphthalene/phenanthrene pathways. Furthermore, the RP007 extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation, but is a member of the recently proposed Class III extradiol dioxygenases. Upstream of the phn catabolic genes are two putative regulatory genes phnR and phnS. phnR is divergently transcribed with respect to the other genes and is similar to the σ⁵⁴-dependent family of positive transcriptional regulators. phnS is a LysR-type transcriptional activator. Expression experiments suggest the phn operon is under regulatory control which may involve PhnR and PhnS.

Two loci encoding aromatic meta cleavage pathways were also cloned from Burkholderia sp. strain RP007. The nucleotide sequence of the catechol 2,3-dioxygenase genes, and adjacent open reading frames, suggest these are meta pathways of different classes which specify catabolism of different aromatic substrates. One of these may be the lower pathway for phenanthrene and naphthalene degradation.

The presence of catabolic genes homologous to the phnAc gene of Burkholderia sp. strain RP007, relative to the archetypal nahAc gene of Pseudomonas putida G7, was screened for in both culturable and nonculturable microbial communities. Of 77 environmental isolates cultured for their ability to degrade naphthalene or phenanthrene, 28 showed homology to nahAc, whilst none of the strains possessed catabolic genes similar to phnAc. However, using phnAc-specific primers, PCR products homologous to phnAc were amplified from DNA extracts of eight hydrocarbon-contaminated soils, demonstrating the widespread distribution of this genotype. Analysis of these phnAc PCR products by nucleotide sequencing of selected cloned products revealed very high (98.5-100%) homology to the phnAc gene of RP007, suggesting the phn genotype is highly conserved. PCR products were also amplified from soil DNA using nahAc-specific primers, and sequencing of these products revealed a diversity similar to that amongst previously described nah-like genes.

year: 1998

title: Researching collaborative artistic practice.

authors: Barbour, Karen

subjects: arts research

abstract: In this paper we offer discussion of collaboration in artistic practice, based on a two-and-a-half-year-long research project undertaken by artists/researchers at the University of Waikato, working in collaboration with local performers. Grounded in kaupapa M¯ori, feminist and phenomenological research methodologies, this research project provided a context for exploring existing understandings of collaborative processes in the arts, and for immersion in and development of alternative processes, across artistic mediums and cultures. Drawing on contemporary understandings of cross-cultural and intercultural practices in the arts, we discuss how shared conceptualisation of ideas, immersion in different creative processes, personal reflection and development over extended periods of time were found to foster collaboration. In this paper we will explore the value and nature of relationships within collaboration, and discuss how selfdetermination or tino rangatiratanga might be maintained within the context of collaborative performance art.

text: Researching collaborative artistic practice. In this paper we offer discussion of collaboration in artistic practice, based on a two-and-a-half-year-long research project undertaken by artists/researchers at the University of Waikato, working in collaboration with local performers. Grounded in kaupapa M¯ori, feminist and phenomenological research methodologies, this research project provided a context for exploring existing understandings of collaborative processes in the arts, and for immersion in and development of alternative processes, across artistic mediums and cultures. Drawing on contemporary understandings of cross-cultural and intercultural practices in the arts, we discuss how shared conceptualisation of ideas, immersion in different creative processes, personal reflection and development over extended periods of time were found to foster collaboration. In this paper we will explore the value and nature of relationships within collaboration, and discuss how selfdetermination or tino rangatiratanga might be maintained within the context of collaborative performance art.

year: 2007