title: A comparative proteomics approach to studying skeletal muscle mitochondria from myostatin knockout mice

authors: Puddick, Jonathan

subjects: mitochondria

abstract: Myostatin is a negative regulator of muscle growth. When it is not present or non-functional double-muscling occurs, the primary characteristic of this phenotype being an increase in muscle mass. Another characteristic of double-muscling is an increased proportion of type IIB muscle fibres, which rely on glycolysis as their primary energy source, as opposed to type IIA and type I fibres which rely on oxidative phosphorylation. This switch in muscle metabolism directly impacts on the mitochondria, as mitochondria from glycolytic muscle fibres have been shown to have differences in metabolic activity. The increased proportion of glycolytic muscle fibres present in myostatin knockout animals provides a unique model to investigate alterations in muscle fibre type metabolism. 

The mouse model of myostatin knockout utilised during this study was generated by genetic deletion of exon three of the myostatin gene. Verification of this knockout was attempted by western blot analysis, but only the latency associated protein (LAP) was detected. Interestingly, the LAP was barely detectable in the knockout muscle suggesting deletion of exon three affects binding of anti-myostatin antibodies to the LAP, as that part of the gene is not deleted. 

A comparison of the basal mitochondrial stress levels was made, also by western blot analysis. The knockout mitochondria showed no change in levels of heat shock protein 60 or superoxide dismutase 2, indicating that they are not being subjected to any increased stress due to the myostatin knockout phenotype. 

A comparative proteomics approach was used to detect changes in the mitochondrial proteome of myostatin knockout gastrocnemius muscle to gain clues to how mitochondria from glycolytic muscle fibres differ from those present in oxidative fibres. This was undertaken using two-dimensional electrophoresis (2-DE), in-gel tryptic digests and peptide mass fingerprinting by mass spectrometry. A 2-DE gel protein loading of 220 g was shown to give the best protein spot resolution and the most crucial step in the loading process was found to be the laying of the immobilized pH gradient, which had to be performed very carefully to obtain a consistent loading pattern. This study resolved only around 160 protein spots out of the estimated 1,000 to 2,000 proteins present in the mitochondria. Modulation of six proteins was seen at a plt0.1 level, but were unable to be identified using the current methodology. More abundant mitochondrial proteins were able to be identified, but showed no significant modulation. Malate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase, which were identified during this study, have been reported to have decreased activity in mitochondria from glycolytic muscle fibres. This study suggests that the change in activity observed by other researchers is due to inhibition of these enzymes in the glycolytic fibres or activation in the oxidative fibres.

text: A comparative proteomics approach to studying skeletal muscle mitochondria from myostatin knockout mice Myostatin is a negative regulator of muscle growth. When it is not present or non-functional double-muscling occurs, the primary characteristic of this phenotype being an increase in muscle mass. Another characteristic of double-muscling is an increased proportion of type IIB muscle fibres, which rely on glycolysis as their primary energy source, as opposed to type IIA and type I fibres which rely on oxidative phosphorylation. This switch in muscle metabolism directly impacts on the mitochondria, as mitochondria from glycolytic muscle fibres have been shown to have differences in metabolic activity. The increased proportion of glycolytic muscle fibres present in myostatin knockout animals provides a unique model to investigate alterations in muscle fibre type metabolism. 

The mouse model of myostatin knockout utilised during this study was generated by genetic deletion of exon three of the myostatin gene. Verification of this knockout was attempted by western blot analysis, but only the latency associated protein (LAP) was detected. Interestingly, the LAP was barely detectable in the knockout muscle suggesting deletion of exon three affects binding of anti-myostatin antibodies to the LAP, as that part of the gene is not deleted. 

A comparison of the basal mitochondrial stress levels was made, also by western blot analysis. The knockout mitochondria showed no change in levels of heat shock protein 60 or superoxide dismutase 2, indicating that they are not being subjected to any increased stress due to the myostatin knockout phenotype. 

A comparative proteomics approach was used to detect changes in the mitochondrial proteome of myostatin knockout gastrocnemius muscle to gain clues to how mitochondria from glycolytic muscle fibres differ from those present in oxidative fibres. This was undertaken using two-dimensional electrophoresis (2-DE), in-gel tryptic digests and peptide mass fingerprinting by mass spectrometry. A 2-DE gel protein loading of 220 g was shown to give the best protein spot resolution and the most crucial step in the loading process was found to be the laying of the immobilized pH gradient, which had to be performed very carefully to obtain a consistent loading pattern. This study resolved only around 160 protein spots out of the estimated 1,000 to 2,000 proteins present in the mitochondria. Modulation of six proteins was seen at a plt0.1 level, but were unable to be identified using the current methodology. More abundant mitochondrial proteins were able to be identified, but showed no significant modulation. Malate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase, which were identified during this study, have been reported to have decreased activity in mitochondria from glycolytic muscle fibres. This study suggests that the change in activity observed by other researchers is due to inhibition of these enzymes in the glycolytic fibres or activation in the oxidative fibres.

year: 2006

title: OzCHI'96 Workshop on the Next Generation of CSCW Systems

authors: Grundy, John C.

subjects: computer science

abstract: This is the Proceedings of the OZCHI'96 Workshop on the Next Generation of CSCW Systems. Thanks must go to Andy Cockburn for inspiring the name of the workshop and thus giving it a (general) theme! The idea for this workshop grew out of discussions with John Venable concerning the Next Generation of CASE Tools workshop which he'd attended in 1995 and 1996. With CSCW research becoming more prominent within the CHI community in Australasia, it seemed a good opportunity to get people together at OZCHI'96 who share this interest. Focusing the workshop on next-generation CSCW system issues produced paper submissions which explored very diverse areas of CSCW, but which all share a common thread of "Where do we go from here?", and, perhaps even more importantly "Why should be doing this?".

text: OzCHI'96 Workshop on the Next Generation of CSCW Systems This is the Proceedings of the OZCHI'96 Workshop on the Next Generation of CSCW Systems. Thanks must go to Andy Cockburn for inspiring the name of the workshop and thus giving it a (general) theme! The idea for this workshop grew out of discussions with John Venable concerning the Next Generation of CASE Tools workshop which he'd attended in 1995 and 1996. With CSCW research becoming more prominent within the CHI community in Australasia, it seemed a good opportunity to get people together at OZCHI'96 who share this interest. Focusing the workshop on next-generation CSCW system issues produced paper submissions which explored very diverse areas of CSCW, but which all share a common thread of "Where do we go from here?", and, perhaps even more importantly "Why should be doing this?".

year: 1996-11-25

title: The Politics of Knowledge and the Reciprocity Gap in the Governance of Intellectual Property Rights

authors: Emett, Raewyn Anne

subjects: Knowledge

abstract: ABSTRACT
This study examines the politics of knowledge benefit-sharing within the re-regulatory framework of the Trade-related Intellectual Property Rights (TRIPS) Agreement which entered into force in 1995 under the auspices of the World Trade Organisation (WTO). The thesis argues that TRIPS both represents a mainstream legal mechanism for states and organisations to govern ideas through trade, and is characterised by a commercial direction away from multilateralism to bilateralism. In its post-implementation phase, this situation has seen the strongest states and corporations consolidate extensive markets in knowledge goods and services. Through analyses of the various levels of international and national governance within the competitive knowledge structure of international political economy (IPE), this study argues that the politicisation of intellectual property has resulted in the dislocation of reciprocity from its normative roots in fairness and trade equity. In conducting this enquiry the research focuses on the political manifestations of intellectual property consistent with long-standing epistemic considerations of reciprocity to test the extent to which the intrinsic public good value of knowledge and its importance to human societies can be reconciled with the privatisation of public forms of knowledge related to discoveries and innovations. 

This thesis draws on Becker's virtue-theoretic model of reciprocity premised on normative obligations to social life to ground its claim that an absence of substantive reciprocal requirements capable of sustaining equivalent returns and rewards is detrimental, both theoretically and practically, to the intrinsic socio-cultural foundation and public good value of knowledge. The conceptual framework of reciprocity defined and developed in this study challenges the materialist controlling authority and proprietary ownership vested in intellectual property law. A new conceptual approach proposed through reciprocity, and provoked by on-going debates about IP recognition, knowledge protection, access and distribution is advanced to counter strengthened and expanded IPRs. Theories of knowledge and property drawn from political philosophies are employed to test whether reciprocity is sufficiently robust enough, or even capable of, encompassing the gap between capital and applied science. This thesis argues that hyper-capitalism at global, national and local levels, accompanied by the boundless accumulation of technology, closes down competition both compromising IP as private rights and the viability of their governance. The political implications of the protection and enforcement of private rights through IP is examined in two key chapters utilising empirical data in relation to traditional knowledge (TK) and reciprocity; the first sets the parameters of TK and the second explores aspects of Māori knowledge systems and reciprocity directed at identifying national and local issues of significance to the debates on IP governance. As a viable direction for knowledge governance this thesis concludes that the gap between the re-regulatory trade framework of intellectual property on the one hand, and reciprocity on the other, requires closing to ameliorate the detrimental disruptions to democratic integrity, fairness and trade equity for significant numbers of communities and peoples around the world.

text: The Politics of Knowledge and the Reciprocity Gap in the Governance of Intellectual Property Rights ABSTRACT
This study examines the politics of knowledge benefit-sharing within the re-regulatory framework of the Trade-related Intellectual Property Rights (TRIPS) Agreement which entered into force in 1995 under the auspices of the World Trade Organisation (WTO). The thesis argues that TRIPS both represents a mainstream legal mechanism for states and organisations to govern ideas through trade, and is characterised by a commercial direction away from multilateralism to bilateralism. In its post-implementation phase, this situation has seen the strongest states and corporations consolidate extensive markets in knowledge goods and services. Through analyses of the various levels of international and national governance within the competitive knowledge structure of international political economy (IPE), this study argues that the politicisation of intellectual property has resulted in the dislocation of reciprocity from its normative roots in fairness and trade equity. In conducting this enquiry the research focuses on the political manifestations of intellectual property consistent with long-standing epistemic considerations of reciprocity to test the extent to which the intrinsic public good value of knowledge and its importance to human societies can be reconciled with the privatisation of public forms of knowledge related to discoveries and innovations. 

This thesis draws on Becker's virtue-theoretic model of reciprocity premised on normative obligations to social life to ground its claim that an absence of substantive reciprocal requirements capable of sustaining equivalent returns and rewards is detrimental, both theoretically and practically, to the intrinsic socio-cultural foundation and public good value of knowledge. The conceptual framework of reciprocity defined and developed in this study challenges the materialist controlling authority and proprietary ownership vested in intellectual property law. A new conceptual approach proposed through reciprocity, and provoked by on-going debates about IP recognition, knowledge protection, access and distribution is advanced to counter strengthened and expanded IPRs. Theories of knowledge and property drawn from political philosophies are employed to test whether reciprocity is sufficiently robust enough, or even capable of, encompassing the gap between capital and applied science. This thesis argues that hyper-capitalism at global, national and local levels, accompanied by the boundless accumulation of technology, closes down competition both compromising IP as private rights and the viability of their governance. The political implications of the protection and enforcement of private rights through IP is examined in two key chapters utilising empirical data in relation to traditional knowledge (TK) and reciprocity; the first sets the parameters of TK and the second explores aspects of Māori knowledge systems and reciprocity directed at identifying national and local issues of significance to the debates on IP governance. As a viable direction for knowledge governance this thesis concludes that the gap between the re-regulatory trade framework of intellectual property on the one hand, and reciprocity on the other, requires closing to ameliorate the detrimental disruptions to democratic integrity, fairness and trade equity for significant numbers of communities and peoples around the world.

year: 2007