title: The Effect of Pax3 Over-expression on Myoblast Function

authors: Brijs, Erik

subjects: Pax3 over-expression

abstract: The paired-box transcription factor 3 (Pax3), is a powerful myogenic regulatory factor during embryo-myogenesis. The expression of Pax3 is involved in determining somitic cell fate, resulting in the formation of the first differentiated muscle, the myotome. In addition, Pax3 expression in embryonic hypaxial precursor cells prevents premature differentiation, whilst promoting cellular migration and thus ensuring the proper development of limb muscles. Subsequent muscle growth in the developing embryo is provided by myogenic precursor cells marked by the expression of Pax3 and its paralogue, Pax7. Following foetal development, these precursor cells establish the distinct lineage of myogenic stem cells referred to as satellite cells, responsible for post-natal development and regeneration. However, satellite cells are not the only population of cells that generate myogenic precursor cells in adult muscle for regeneration and growth. Recently, it has been shown that in adult muscle, Pax3 is expressed in a population of novel, interstitial cells, and is up-regulated upon muscle injury.
To determine the role played by Pax3 in post-natal muscle development and regeneration, three Pax3 over-expressing myoblast (C2C12) cell lines were generated and experimentally compared with control cells. Four facets of myoblast function were assessed: proliferation, differentiation, survival and migration. 
The proliferation assay showed that Pax3 over-expression significantly (Plt0.001) slowed C2C12 myoblast proliferation, Pax3 over-expressing clones only reached 63% of the proliferative rate of control C2C12 cells. Furthermore, this study clearly showed that over-expression of Pax3 significantly (Plt0.01) delayed the differentiation of C2C12 myoblasts. Analysis showed that following 72 h of low serum induced differentiation 52% of the control C2C12 cell population had fused to form myofibres, in comparison only 16-32% of the myoblast population that over-expressed Pax3 had fused to from myofibres. Taken together, these data suggest that following satellite cell activation, Pax3 expression could be maintained in precursor cells to expand the myogenic population, as their myogenic commitment is delayed by Pax3 expression and this would facilitate increased cell cycling, albeit at a reduced rate. Furthermore, a low percentage of fusion strongly indicates a higher proportion of cells that are renewing the quiescent satellite cell population.
Furthermore, over-expression of Pax3 also conferred a dose-dependent anti-apoptotic function to C2C12 myoblasts. Myoblasts that over-expressed a low level of Pax3 were more resistant to apoptosis in comparison to control C2C12 cells and to myoblasts that over-expressed Pax3 at higher level. The anti-apoptotic function conferred by Pax3 expression would come into effect within the Pax3+ self-renewing satellite cell lineage. 
Finally, Pax3 over-expression was shown to increase the migratory ability of C2C12 myoblasts. In a chemotaxis assay, there was a strong indication that an intermediate over-expression of Pax3 increased myoblast sensitivity to chemotactic signals, priming cells for migration.  The delay in differentiation also exhibited by Pax3 over-expression may function to allow satellite cell-derived progenitors to migrate to sites of injury, therefore enhancing skeletal muscle regenerative capacity.

text: The Effect of Pax3 Over-expression on Myoblast Function The paired-box transcription factor 3 (Pax3), is a powerful myogenic regulatory factor during embryo-myogenesis. The expression of Pax3 is involved in determining somitic cell fate, resulting in the formation of the first differentiated muscle, the myotome. In addition, Pax3 expression in embryonic hypaxial precursor cells prevents premature differentiation, whilst promoting cellular migration and thus ensuring the proper development of limb muscles. Subsequent muscle growth in the developing embryo is provided by myogenic precursor cells marked by the expression of Pax3 and its paralogue, Pax7. Following foetal development, these precursor cells establish the distinct lineage of myogenic stem cells referred to as satellite cells, responsible for post-natal development and regeneration. However, satellite cells are not the only population of cells that generate myogenic precursor cells in adult muscle for regeneration and growth. Recently, it has been shown that in adult muscle, Pax3 is expressed in a population of novel, interstitial cells, and is up-regulated upon muscle injury.
To determine the role played by Pax3 in post-natal muscle development and regeneration, three Pax3 over-expressing myoblast (C2C12) cell lines were generated and experimentally compared with control cells. Four facets of myoblast function were assessed: proliferation, differentiation, survival and migration. 
The proliferation assay showed that Pax3 over-expression significantly (Plt0.001) slowed C2C12 myoblast proliferation, Pax3 over-expressing clones only reached 63% of the proliferative rate of control C2C12 cells. Furthermore, this study clearly showed that over-expression of Pax3 significantly (Plt0.01) delayed the differentiation of C2C12 myoblasts. Analysis showed that following 72 h of low serum induced differentiation 52% of the control C2C12 cell population had fused to form myofibres, in comparison only 16-32% of the myoblast population that over-expressed Pax3 had fused to from myofibres. Taken together, these data suggest that following satellite cell activation, Pax3 expression could be maintained in precursor cells to expand the myogenic population, as their myogenic commitment is delayed by Pax3 expression and this would facilitate increased cell cycling, albeit at a reduced rate. Furthermore, a low percentage of fusion strongly indicates a higher proportion of cells that are renewing the quiescent satellite cell population.
Furthermore, over-expression of Pax3 also conferred a dose-dependent anti-apoptotic function to C2C12 myoblasts. Myoblasts that over-expressed a low level of Pax3 were more resistant to apoptosis in comparison to control C2C12 cells and to myoblasts that over-expressed Pax3 at higher level. The anti-apoptotic function conferred by Pax3 expression would come into effect within the Pax3+ self-renewing satellite cell lineage. 
Finally, Pax3 over-expression was shown to increase the migratory ability of C2C12 myoblasts. In a chemotaxis assay, there was a strong indication that an intermediate over-expression of Pax3 increased myoblast sensitivity to chemotactic signals, priming cells for migration.  The delay in differentiation also exhibited by Pax3 over-expression may function to allow satellite cell-derived progenitors to migrate to sites of injury, therefore enhancing skeletal muscle regenerative capacity.

year: 2009

title: The concept of income within the New Zealand taxation system

authors: Alley, Clinton

subjects: accounting

abstract: The paper looks at the core provisions used in the procedure for calculating and satisfying income tax liability as applied by the New Zealand Income Tax Act 2004. This Act has introduced an additional part to the subpart BC flowchart and a new term “non-residents’ foreign-sourced income’ to assist in the determination of income. The authors review these additions to the Income Tax Act and suggest that further improvements can be made. To this end they include proposed amendments to Part B ( including the subpart BC flowchart) and Part C of ITA 2004

text: The concept of income within the New Zealand taxation system The paper looks at the core provisions used in the procedure for calculating and satisfying income tax liability as applied by the New Zealand Income Tax Act 2004. This Act has introduced an additional part to the subpart BC flowchart and a new term “non-residents’ foreign-sourced income’ to assist in the determination of income. The authors review these additions to the Income Tax Act and suggest that further improvements can be made. To this end they include proposed amendments to Part B ( including the subpart BC flowchart) and Part C of ITA 2004

year: 2006-09

title: Te Kāuta

authors: Roa, Raukura

subjects: Kōrero paki

abstract: He rite tonu tā mātou hokihoki ki te take o Pirongia maunga, ki te tutungi i te ahi kā i Pūrekireki Marae. Ia wā ka hoki mātou ki tēnei o ngā marae, ka hīkaka taku ngākau, i te mea, ko taku mahi i reira, he tiaki ahi. Ao noa, pō noa, ko au te kaiwhakatutungi i te ahi o te kāuta, kei kewa. Ko te ahi o te kāuta o Pūrekireki marae, he ahi nui, he ahi kaitā. E ono tāngata pea ka uru ki te rua o te ahi nei. Nō reira, ko taku mahi i te rā, he kohi rākau i waho, he tapahi wahie, he whakamaroke anō hoki i te wahie, inā mākū i te ua. I ngā pō, kia hoki rā anō te iwi ki te moe, ko tāku, he noho ki roto i te kāuta me te tutungi ahi, kia wera tonu ai te wai mō āpōpō. Kei pōhēhē mai, he mahi mokemoke tēnei! Ehara! Ia pō, ka noho mai aku kuia ki taku taha ki te pahupahu. He nui ngā akoranga me ngā mātauranga i tuku iho mai ki a au i roto i tērā kāuta, i te taha o tērā ahi. Me uaua ka kitea tēnei momo ahi kaitā i ngā marae i ēnei rā. Kāore e whakaaengia ana e ngā ture Pākehā ngā ahi kaitā, pēnei nei te rahi, i roto i ngā kāuta o ngā marae; ‘Health and Safety Standards’ tā rātou i whakatau ai. Nō reira, i karangangia e taku pāpā tētehi hui ki te marae kia wānangahia te whakahōutanga o tō mātou kāuta. Nō taku rongotanga ake i te kaupapa o tēnei hui, ka tangi taku ngākau ki tō mātou kāuta. Nō reira, i whakarite ahau kia haere i tana taha.

text: Te Kāuta He rite tonu tā mātou hokihoki ki te take o Pirongia maunga, ki te tutungi i te ahi kā i Pūrekireki Marae. Ia wā ka hoki mātou ki tēnei o ngā marae, ka hīkaka taku ngākau, i te mea, ko taku mahi i reira, he tiaki ahi. Ao noa, pō noa, ko au te kaiwhakatutungi i te ahi o te kāuta, kei kewa. Ko te ahi o te kāuta o Pūrekireki marae, he ahi nui, he ahi kaitā. E ono tāngata pea ka uru ki te rua o te ahi nei. Nō reira, ko taku mahi i te rā, he kohi rākau i waho, he tapahi wahie, he whakamaroke anō hoki i te wahie, inā mākū i te ua. I ngā pō, kia hoki rā anō te iwi ki te moe, ko tāku, he noho ki roto i te kāuta me te tutungi ahi, kia wera tonu ai te wai mō āpōpō. Kei pōhēhē mai, he mahi mokemoke tēnei! Ehara! Ia pō, ka noho mai aku kuia ki taku taha ki te pahupahu. He nui ngā akoranga me ngā mātauranga i tuku iho mai ki a au i roto i tērā kāuta, i te taha o tērā ahi. Me uaua ka kitea tēnei momo ahi kaitā i ngā marae i ēnei rā. Kāore e whakaaengia ana e ngā ture Pākehā ngā ahi kaitā, pēnei nei te rahi, i roto i ngā kāuta o ngā marae; ‘Health and Safety Standards’ tā rātou i whakatau ai. Nō reira, i karangangia e taku pāpā tētehi hui ki te marae kia wānangahia te whakahōutanga o tō mātou kāuta. Nō taku rongotanga ake i te kaupapa o tēnei hui, ka tangi taku ngākau ki tō mātou kāuta. Nō reira, i whakarite ahau kia haere i tana taha.

year: 2016