title: Prolactin-induced proteins and dependence on protein synthesis for β-casein expression in mouse mammary cells

authors: Beaton, Angela

abstract: The regulation of milk protein gene expression in mammary epithelial cells occurs in response to multiple hormonal signals, plus cell-matrix and cell-cell interactions. A number of signalling molecules have been implicated in this process, but the exact molecular mechanisms regulating milk protein gene expression remain to be established. The project described in this thesis focuses on investigating the de novo synthesis of proteins in response to prolactin in mammary epithelial cells, to determine the role of de novo synthesis in lactogenic signalling, and to identify proteins which are induced by prolactin that could act as mediators of lactogenic signals. Two main approaches were taken:

(1) The results of proteome analyses using two-dimensional electrophoresis revealed a number of prolactin-responsive proteins in cells from the mouse mammary epithelial cell line, COMMA-D, and also lactation-associated proteins in freshly isolated mouse mammary epithelial cells. Two prolactin-induced and lactation-associated proteins, p77 and p63, were identified as glucose regulated protein 78 (grp78) and protein disulphide isomerase (PDI), and assessed as potential regulators of lactation. These proteins are reticuloplasmins and probably function in mammary epithelial cells in the processing of milk proteins for secretion. The results of this investigation have shown that grp78 and PDI were not rapidly induced in response to prolactin, thus it is unlikely that these proteins are involved in mediating lactogenic signals in response to prolactin.

(2) The requirement for de novo protein synthesis for maximal prolactin-stimulated β-casein gene expression was evaluated in COMMA-D cells using the protein synthesis inhibitors cycloheximide (CHX) and anisomycin. It appears that protein synthesis is required at the level of transcription for maximal induction and maintenance of β-casein gene expression (over the first 8 h of prolactin treatment). Furthermore, this effect was shown to be very rapid and independent of the lactogenic hormones, insulin and hydrocortisone, and the extracellular matrix. To determine the molecular basis for the effect of CHX on β-casein mRNA levels, STAT5 protein levels and DNA-binding activity were analysed in COMMA-D cells cultured in the presence or absence of prolactin and/or CHX. The results of Western analyses showed that STAT5 protein levels were not significantly altered in  the presence or absence of (prolactin and/ or) CHX. A DNA probe, denoted STAT5(30), derived from the rat β-casein promoter sequence (-104 to -75), which contains a high affinity STAT5 consensus sequence was used for EMSA. CHX treatment stimulated the induction of a DNA-binding activity of distinct mobility to STAT5. This protein-DNA complex was shown to contain the p50 subunit of NF-κB. Detailed analysis revealed that CHX-stimulated NF-κB bound to a putative κB half-site on the STAT5(30) probe (GGAATT), which overlaps the high-affinity STAT5 DNA-binding site (TTCTTGGAA). Therefore, the effect of CHX appears to be mediated by inhibition of IκB synthesis, leading to the activation of NF-κB. Since the κB site overlaps the STAT5 binding site, it is possible that NF-κB competes for binding to the β-casein promoter and sterically hinders STAT5 binding and transactivation of β-casein transcription. It is conceivable that competition between NF-κB and STAT5 is functionally significant if NF-κB is activated in response to physiological signals in the mammary gland. This remains to be demonstrated.

text: Prolactin-induced proteins and dependence on protein synthesis for β-casein expression in mouse mammary cells The regulation of milk protein gene expression in mammary epithelial cells occurs in response to multiple hormonal signals, plus cell-matrix and cell-cell interactions. A number of signalling molecules have been implicated in this process, but the exact molecular mechanisms regulating milk protein gene expression remain to be established. The project described in this thesis focuses on investigating the de novo synthesis of proteins in response to prolactin in mammary epithelial cells, to determine the role of de novo synthesis in lactogenic signalling, and to identify proteins which are induced by prolactin that could act as mediators of lactogenic signals. Two main approaches were taken:

(1) The results of proteome analyses using two-dimensional electrophoresis revealed a number of prolactin-responsive proteins in cells from the mouse mammary epithelial cell line, COMMA-D, and also lactation-associated proteins in freshly isolated mouse mammary epithelial cells. Two prolactin-induced and lactation-associated proteins, p77 and p63, were identified as glucose regulated protein 78 (grp78) and protein disulphide isomerase (PDI), and assessed as potential regulators of lactation. These proteins are reticuloplasmins and probably function in mammary epithelial cells in the processing of milk proteins for secretion. The results of this investigation have shown that grp78 and PDI were not rapidly induced in response to prolactin, thus it is unlikely that these proteins are involved in mediating lactogenic signals in response to prolactin.

(2) The requirement for de novo protein synthesis for maximal prolactin-stimulated β-casein gene expression was evaluated in COMMA-D cells using the protein synthesis inhibitors cycloheximide (CHX) and anisomycin. It appears that protein synthesis is required at the level of transcription for maximal induction and maintenance of β-casein gene expression (over the first 8 h of prolactin treatment). Furthermore, this effect was shown to be very rapid and independent of the lactogenic hormones, insulin and hydrocortisone, and the extracellular matrix. To determine the molecular basis for the effect of CHX on β-casein mRNA levels, STAT5 protein levels and DNA-binding activity were analysed in COMMA-D cells cultured in the presence or absence of prolactin and/or CHX. The results of Western analyses showed that STAT5 protein levels were not significantly altered in  the presence or absence of (prolactin and/ or) CHX. A DNA probe, denoted STAT5(30), derived from the rat β-casein promoter sequence (-104 to -75), which contains a high affinity STAT5 consensus sequence was used for EMSA. CHX treatment stimulated the induction of a DNA-binding activity of distinct mobility to STAT5. This protein-DNA complex was shown to contain the p50 subunit of NF-κB. Detailed analysis revealed that CHX-stimulated NF-κB bound to a putative κB half-site on the STAT5(30) probe (GGAATT), which overlaps the high-affinity STAT5 DNA-binding site (TTCTTGGAA). Therefore, the effect of CHX appears to be mediated by inhibition of IκB synthesis, leading to the activation of NF-κB. Since the κB site overlaps the STAT5 binding site, it is possible that NF-κB competes for binding to the β-casein promoter and sterically hinders STAT5 binding and transactivation of β-casein transcription. It is conceivable that competition between NF-κB and STAT5 is functionally significant if NF-κB is activated in response to physiological signals in the mammary gland. This remains to be demonstrated.

year: 1998

title: Mitochondrial DNA (COI) analyses reveal that amphipod diversity is associated with environmental heterogeneity in deep-sea habitats

authors: Knox, Matthew Andrew

subjects: Biodiversity

abstract: The relationship between species diversity and environmental parameters is poorly understood for the mobile macrofauna of deep-sea habitats due to under-sampling and subsequent lack of accurate taxonomic information. To redress this, cytochrome oxidase c subunit I (COI) DNA sequences were used to estimate species diversity and to compare phoxocephalid amphipod assemblages among 20 stations encompassing a range of environmental conditions. Two regions, east (Chatham Rise) and west (Challenger Plateau) of New Zealand were sampled to depths of 200–1200 m with an epibenthic sled. Using a comparison among identified morphospecies, we found a clear gap in sequence divergences between 6% and 13% and used a 6% threshold to designate molecular operational taxonomic units (MOTUs), as a surrogate to putative species. DNA sequences (n = 297) revealed high total diversity (n = 49 MOTUs), as well as high beta diversity (28 MOTUs found at single location only). Novel phoxocephalid MOTUs were found at most stations, especially on Challenger Plateau and the flanks of Chatham Rise. Analyses of interstation assemblages revealed a major split between regions, indicating minimal overlap in taxon distributions. A cluster of highly similar stations was identified, broadly distributed over the crest of Chatham Rise, in association with elevated food availability, probably resulting from higher surface productivity and relatively shallow depth. Accordingly, multivariate analysis revealed a strong correlation between phoxocephalid assemblages and food supply. This study highlights the value of molecular approaches, in particular COI sequences, for quantifying and comparing diversity in under-sampled and/or under-studied taxa.

text: Mitochondrial DNA (COI) analyses reveal that amphipod diversity is associated with environmental heterogeneity in deep-sea habitats The relationship between species diversity and environmental parameters is poorly understood for the mobile macrofauna of deep-sea habitats due to under-sampling and subsequent lack of accurate taxonomic information. To redress this, cytochrome oxidase c subunit I (COI) DNA sequences were used to estimate species diversity and to compare phoxocephalid amphipod assemblages among 20 stations encompassing a range of environmental conditions. Two regions, east (Chatham Rise) and west (Challenger Plateau) of New Zealand were sampled to depths of 200–1200 m with an epibenthic sled. Using a comparison among identified morphospecies, we found a clear gap in sequence divergences between 6% and 13% and used a 6% threshold to designate molecular operational taxonomic units (MOTUs), as a surrogate to putative species. DNA sequences (n = 297) revealed high total diversity (n = 49 MOTUs), as well as high beta diversity (28 MOTUs found at single location only). Novel phoxocephalid MOTUs were found at most stations, especially on Challenger Plateau and the flanks of Chatham Rise. Analyses of interstation assemblages revealed a major split between regions, indicating minimal overlap in taxon distributions. A cluster of highly similar stations was identified, broadly distributed over the crest of Chatham Rise, in association with elevated food availability, probably resulting from higher surface productivity and relatively shallow depth. Accordingly, multivariate analysis revealed a strong correlation between phoxocephalid assemblages and food supply. This study highlights the value of molecular approaches, in particular COI sequences, for quantifying and comparing diversity in under-sampled and/or under-studied taxa.

year: 2012

title: He pūrongo whakahokinga kōrero a te akomanga rūmaki o Te Tohu Paetahi

authors: Nock, Sophie

subjects: Reo Māori

abstract: Ko te whāinga matua o tēnei rangahau he whakakaokao, he wetewete āno hoki i ngā whakahokinga kōrero a te hunga uiui i kake atu ai ki runga i te waka o Te Tohu Paetahi i te tau 2008, i raro i te kōawa o Rehutai, i raro anō hoki i te maru o Te Whare Wānanga o Waikato. Ka aro atu ki ngā urupare a te hunga tekau mā ono mō te kaupapa nei o Te Tohu Paetahi, ka mutu, ka matapakihia ngā kōrero me ngā whakaaro o taua hunga kia āta kitea ai, āe rānei e tika ana te whakahaere, te mahi whakaako i te reo, te tautoko mai hoki a ngā kaiwhakaako me te kura. I te mutunga iho tautoko katoa te hunga uiui ki aua mea katoa.

text: He pūrongo whakahokinga kōrero a te akomanga rūmaki o Te Tohu Paetahi Ko te whāinga matua o tēnei rangahau he whakakaokao, he wetewete āno hoki i ngā whakahokinga kōrero a te hunga uiui i kake atu ai ki runga i te waka o Te Tohu Paetahi i te tau 2008, i raro i te kōawa o Rehutai, i raro anō hoki i te maru o Te Whare Wānanga o Waikato. Ka aro atu ki ngā urupare a te hunga tekau mā ono mō te kaupapa nei o Te Tohu Paetahi, ka mutu, ka matapakihia ngā kōrero me ngā whakaaro o taua hunga kia āta kitea ai, āe rānei e tika ana te whakahaere, te mahi whakaako i te reo, te tautoko mai hoki a ngā kaiwhakaako me te kura. I te mutunga iho tautoko katoa te hunga uiui ki aua mea katoa.

year: 2012-05