title: Purification and properties of an aryl beta-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli.

authors: Hudson, R.C.

subjects: beta-xylosidase

abstract: An aryl beta-xylosidase was purified to homogeneity from an Escherichia coli strain containing a recombinant plasmid carrying a beta-xylosidase (EC 3.2.1.37) gene from the extremely thermophilic anaerobic bacterium isolate Tp8T6.3.3.1 ('Caldocellum saccharolyticum'). It has a pI of 4.3 and shows optimal activity at pH 5.7. The enzyme is highly specific, acting on o- and p-nitrophenyl beta-D-xylopyranosides and minimally on p-nitrophenyl alpha-L-arabinopyranoside. It does not act on xylobiose. The Km for p-nitrophenyl beta-D-xylopyranoside at the optimum pH for activity is 10 mM, and at pH 7.0 is 6.7 mM. Xylose is a competitive inhibitor with Ki 40 mM. Thermal inactivation follows first-order kinetics at 65 and 70 degrees C with t1/2 values of 4.85 h and 40 min respectively. The t1/2 at 70 degrees C is increased 3-fold and 4-fold by the addition of 0.5 mg of BSA/ml and 2 mM-dithiothreitol respectively.

text: Purification and properties of an aryl beta-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli. An aryl beta-xylosidase was purified to homogeneity from an Escherichia coli strain containing a recombinant plasmid carrying a beta-xylosidase (EC 3.2.1.37) gene from the extremely thermophilic anaerobic bacterium isolate Tp8T6.3.3.1 ('Caldocellum saccharolyticum'). It has a pI of 4.3 and shows optimal activity at pH 5.7. The enzyme is highly specific, acting on o- and p-nitrophenyl beta-D-xylopyranosides and minimally on p-nitrophenyl alpha-L-arabinopyranoside. It does not act on xylobiose. The Km for p-nitrophenyl beta-D-xylopyranoside at the optimum pH for activity is 10 mM, and at pH 7.0 is 6.7 mM. Xylose is a competitive inhibitor with Ki 40 mM. Thermal inactivation follows first-order kinetics at 65 and 70 degrees C with t1/2 values of 4.85 h and 40 min respectively. The t1/2 at 70 degrees C is increased 3-fold and 4-fold by the addition of 0.5 mg of BSA/ml and 2 mM-dithiothreitol respectively.

year: 1991

title: The Role of Amino Acids and the Threonine-SAM Pathway in the Development of Bovine Inner Cell Mass and Pluripotency

authors: Najafzadeh, Vahid

subjects: amino acid

abstract: Pluripotent stem cells (PSCs) are the foundation of all cell types in the body including functional gametes. They are derived either from the inner cell mass (ICM) of preimplantation mouse and rat embryos, known as embryonic pluripotent stem cells (ePSCs) or embryonic stem cells (ESCs) or via reprogramming of somatic cells to their pluripotent state, known as induced pluripotent stem cells (iPSCs). PSCs are excellent tools for gene editing and accelerated breeding in livestock animals. So far, germline-competent PSCs have only been isolated in mice and rats. Bovine ESCs are yet to be isolated due to the inadequate understanding of the mechanisms involved in bovine pluripotency. This study aimed to investigate the metabolic aspects of bovine ICM development and pluripotency. 
In this study, amino acids were screened via a targeted depletion approach to determine if any amino acid was critical for bovine ICM development and pluripotency. Experiments were conducted on in vitro fertilised day (D) 5 preimplantation bovine embryos, individually cultured in chemically-defined, protein- and glutamine-free synthetic oviduct fluid with variations of essential (E) and non-essential (NE) amino acids (AAs) until D8. Depleting (Δ) one (threonine, methionine), two (threonine_methionine, cysteine_methionine, cysteine_threonine, isoleucine_leucine, isoleucine_lysine, leucine_lysine), three (cysteine_methionine_threonine, isoleucine_leucine_lysine) or six (histidine_phenylalanine_arginine_valine_tryptophan_tyrosine) EAAs did not affect ICM formation, even when NEAAs were also removed from Δthreonine and Δmethionine groups. However, depleting another six (cysteine_isoleucine_lysine_leucine_methionine_threonine), nine (+cysteine_methionine_threonine, +isoleucine_leucine_lysine), eleven EAAs (+threonine, +methionine) or all twelve EAAs impaired blastocyst development. In vitro fertilised D1 preimplantation mouse embryos cultured from D1-4 were also not affected by a Δthreonine culture condition.
The role of the threonine dehydrogenase (TDH)-mediated threonine-s-adenosylmethionine (SAM) pathway in bovine ICM development and pluripotency in bovine embryos and cells was also investigated. This metabolic pathway has been shown to play a critical role in sustaining mouse pluripotency. TDH expression was probed at mRNA and protein levels, and its activity was knocked down using chemical inhibitors quinazolinecarboxamide (Qc1) and 3-hydroxynorvaline (3-HNV). The effect of TDH inhibition on embryo development, cell numbers, lineage-specific marker gene expression, histone trimethylation and autophagy was studied. 
TDH was present in trophectoderm (TE), ICM and 6 day old (D6) ICM outgrowths but absent in D6 TE primary cultures. In bovine embryos, Qc1 reduced blastocyst grade 1-3 (B¹⁻³) and 
B¹⁻² embryo development 1.5- and 2-fold, respectively, compared to the dimethyl sulfoxide (DMSO) controls. Qc1 also reduced ICM and TE cell numbers ~2-fold. This cell loss was due to increased autophagy. Qc1-treated ICM and TE nuclei were explicitly hypermethylated at histone (H)3 lysine (K)4 trimethylation (me3), while H3K9me3 and H3K27me3 were not affected. Hypoblast (PDGRFα), pluripotency-associated epiblast (NANOG, FGF4, and SOX2) and TE (CDX2) markers were not altered in the Qc1 group compared to DMSO controls. Qc1 also severely compromised mouse embryos cultured from day 1-4. In bovine cells, Qc1 compromised fresh ICM, D6 ICM outgrowths and fresh TE cells but failed to impair D6 TE primary cultures. 3-HNV severely compromised bovine embryo development, D1 ICM outgrowths, D6 TE primary cultures and bovine embryonic fibroblast (BEF) cells. However, it unexpectedly severely impaired TDH-free skin fibroblasts in a Δthreonine culture condition. 
The results indicate that the effect of amino acid starvation on embryos is different from that on individual cells. The findings also suggest that TDH is vital for bovine embryonic development via regulating H3K4me3 levels. Moreover, the results denote that TDH becomes restricted to cultured ICM cells and disappears from TE primary cultures. This may indicate that TDH is essential for maintaining bovine pluripotency. 3-HNV was claimed to be a specific TDH enzyme inhibitor; However, this study showed that it compromises TDH-free cells in a Δthreonine culture condition, suggesting that it does not specifically impair cells and embryos via the TDH-mediated threonine-SAM pathway.

text: The Role of Amino Acids and the Threonine-SAM Pathway in the Development of Bovine Inner Cell Mass and Pluripotency Pluripotent stem cells (PSCs) are the foundation of all cell types in the body including functional gametes. They are derived either from the inner cell mass (ICM) of preimplantation mouse and rat embryos, known as embryonic pluripotent stem cells (ePSCs) or embryonic stem cells (ESCs) or via reprogramming of somatic cells to their pluripotent state, known as induced pluripotent stem cells (iPSCs). PSCs are excellent tools for gene editing and accelerated breeding in livestock animals. So far, germline-competent PSCs have only been isolated in mice and rats. Bovine ESCs are yet to be isolated due to the inadequate understanding of the mechanisms involved in bovine pluripotency. This study aimed to investigate the metabolic aspects of bovine ICM development and pluripotency. 
In this study, amino acids were screened via a targeted depletion approach to determine if any amino acid was critical for bovine ICM development and pluripotency. Experiments were conducted on in vitro fertilised day (D) 5 preimplantation bovine embryos, individually cultured in chemically-defined, protein- and glutamine-free synthetic oviduct fluid with variations of essential (E) and non-essential (NE) amino acids (AAs) until D8. Depleting (Δ) one (threonine, methionine), two (threonine_methionine, cysteine_methionine, cysteine_threonine, isoleucine_leucine, isoleucine_lysine, leucine_lysine), three (cysteine_methionine_threonine, isoleucine_leucine_lysine) or six (histidine_phenylalanine_arginine_valine_tryptophan_tyrosine) EAAs did not affect ICM formation, even when NEAAs were also removed from Δthreonine and Δmethionine groups. However, depleting another six (cysteine_isoleucine_lysine_leucine_methionine_threonine), nine (+cysteine_methionine_threonine, +isoleucine_leucine_lysine), eleven EAAs (+threonine, +methionine) or all twelve EAAs impaired blastocyst development. In vitro fertilised D1 preimplantation mouse embryos cultured from D1-4 were also not affected by a Δthreonine culture condition.
The role of the threonine dehydrogenase (TDH)-mediated threonine-s-adenosylmethionine (SAM) pathway in bovine ICM development and pluripotency in bovine embryos and cells was also investigated. This metabolic pathway has been shown to play a critical role in sustaining mouse pluripotency. TDH expression was probed at mRNA and protein levels, and its activity was knocked down using chemical inhibitors quinazolinecarboxamide (Qc1) and 3-hydroxynorvaline (3-HNV). The effect of TDH inhibition on embryo development, cell numbers, lineage-specific marker gene expression, histone trimethylation and autophagy was studied. 
TDH was present in trophectoderm (TE), ICM and 6 day old (D6) ICM outgrowths but absent in D6 TE primary cultures. In bovine embryos, Qc1 reduced blastocyst grade 1-3 (B¹⁻³) and 
B¹⁻² embryo development 1.5- and 2-fold, respectively, compared to the dimethyl sulfoxide (DMSO) controls. Qc1 also reduced ICM and TE cell numbers ~2-fold. This cell loss was due to increased autophagy. Qc1-treated ICM and TE nuclei were explicitly hypermethylated at histone (H)3 lysine (K)4 trimethylation (me3), while H3K9me3 and H3K27me3 were not affected. Hypoblast (PDGRFα), pluripotency-associated epiblast (NANOG, FGF4, and SOX2) and TE (CDX2) markers were not altered in the Qc1 group compared to DMSO controls. Qc1 also severely compromised mouse embryos cultured from day 1-4. In bovine cells, Qc1 compromised fresh ICM, D6 ICM outgrowths and fresh TE cells but failed to impair D6 TE primary cultures. 3-HNV severely compromised bovine embryo development, D1 ICM outgrowths, D6 TE primary cultures and bovine embryonic fibroblast (BEF) cells. However, it unexpectedly severely impaired TDH-free skin fibroblasts in a Δthreonine culture condition. 
The results indicate that the effect of amino acid starvation on embryos is different from that on individual cells. The findings also suggest that TDH is vital for bovine embryonic development via regulating H3K4me3 levels. Moreover, the results denote that TDH becomes restricted to cultured ICM cells and disappears from TE primary cultures. This may indicate that TDH is essential for maintaining bovine pluripotency. 3-HNV was claimed to be a specific TDH enzyme inhibitor; However, this study showed that it compromises TDH-free cells in a Δthreonine culture condition, suggesting that it does not specifically impair cells and embryos via the TDH-mediated threonine-SAM pathway.

year: 2018

title: Dual cultural identity and tangihanga: Conflict, resolution and unexpected outcomes

authors: Edge, Kiri

subjects: bereavement

abstract: New Zealand has a significant number of dual-cultural whānau (families) which incorporate the identities of both Pākehā (New Zealander of European descent) and Māori (indigenous peoples of New Zealand). Little attention has been paid to the bereavement processes that will inevitably impact upon the lives of these whānau/families. As part of the Tangihanga Research Programme based at The University of Waikato, a directed study was conducted with a participant whose family/whānau included two life ways: Māori and Pākehā. An open-ended narrative approach was used to explore the participant’s bereavement after the death of his beloved wife. Two central themes emerged within the narrative, which related to conflict and eventual resolution. Decision-making processes and language played significant roles in the conflict experienced by the participant. Communication and compromise helped to resolve these conflicts. Unexpected outcomes included new understandings and strengthened connections between the participant and his wife’s marae.

text: Dual cultural identity and tangihanga: Conflict, resolution and unexpected outcomes New Zealand has a significant number of dual-cultural whānau (families) which incorporate the identities of both Pākehā (New Zealander of European descent) and Māori (indigenous peoples of New Zealand). Little attention has been paid to the bereavement processes that will inevitably impact upon the lives of these whānau/families. As part of the Tangihanga Research Programme based at The University of Waikato, a directed study was conducted with a participant whose family/whānau included two life ways: Māori and Pākehā. An open-ended narrative approach was used to explore the participant’s bereavement after the death of his beloved wife. Two central themes emerged within the narrative, which related to conflict and eventual resolution. Decision-making processes and language played significant roles in the conflict experienced by the participant. Communication and compromise helped to resolve these conflicts. Unexpected outcomes included new understandings and strengthened connections between the participant and his wife’s marae.

year: 2010