Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities*

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-b-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsinlike activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin- like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyland radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly- Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3,4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by twodimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities

Design process excludes users : the co-creation activities between user and designer

User needs are a fundamental element of design. If the design process does not properly reflect user needs, the design will be severely compromised. Therefore, it is worthwhile to investigate how the user is, and user needs are, understood in the design process. In this article, three accepted linear process models for web site and interactive media design are reviewed in terms of the designer and user participation. The article then proposes a user-evolving collaborative design process which is built on co-creation activities between designer and user. Co-creation activities across the entire design process structurally and ontologically reposition the users, and user needs, centrally, which allows the designers to holistically approach to the user needs through building a partnership with the users. Co-creation creates an equal evolving participatory process between user and designer towards sharing values and knowledge and creating new domains of collective creativity.

Early years activities for NLNW 2012 - Pearl Barley & Charlie Parsley

This Early Years unit is designed to engage students in a sequence of activities that use a range of literacies which explore feelings, friendships and personality traits. The text, Pearl Barley and Charlie Parsley, is the focus of this unit. Suggested activities promote participation through simple and effective strategies, using technology, process drama, graphic ogranisers and critical literacy. This unit allows for flexibility so teachers can select activities that best suit time, resources and students' needs and interests. Students are asked to make connections between the text and their own friendships, understanding that the best of friends can be different in almost every way.

Combination of MEK-ERK inhibitor and hyaluronic acid has a synergistic effect on anti-hypertrophic and pro-chondrogenic activities in osteoarthritis treatment

We hypothesised that a potentially disease-modifying osteoarthritis (OA) drug such as hyaluronic acid (HA) given in combination with anti-inflammatory signalling agents such as mitogen-activated protein kinase kinase–extracellular signal-regulated kinase (MEK-ERK) signalling inhibitor (U0126) could result in additive or synergistic effects on preventing the degeneration of articular cartilage. Chondrocyte differentiation and hypertrophy were evaluated using human OA primary cells treated with either HA or U0126, or the combination of HA + U0126. Cartilage degeneration in menisectomy (MSX) induced rat OA model was investigated by intra-articular delivery of either HA or U0126, or the combination of HA + U0126. Histology, immunostaining, RT-qPCR, Western blotting and zymography were performed to assess the expression of cartilage matrix proteins and hypertrophic markers. Phosphorylated ERK (pERK)1/2-positive chondrocytes were significantly higher in OA samples compared with those in healthy control suggesting the pathological role of that pathway in OA. It was noted that HA + U0126 significantly reduced the levels of pERK, chondrocyte hypertrophic markers (COL10 and RUNX2) and degenerative markers (ADAMTs5 and MMP-13), however, increased the levels of chondrogenic markers (COL2) compared to untreated or the application of HA or U0126 alone. In agreement with the results in vitro, intra-articular delivery of HA + U0126 showed significant therapeutic improvement of cartilage in rat MSX OA model compared with untreated or the application of HA or U0126 alone. Our study suggests that the combination of HA and MEK-ERK inhibition has a synergistic effect on preventing cartilage degeneration.

Gaps in organizational leadership : linking strategic and operational activities through design-led propositions

The relationship between design process and business systems has been of interest to both practitioners and researchers exploring the numerous opportunities and challenges of this unlikely relationship. Often the relationship is presented as building design thinking capability within an organization, which can be broadly described as the union of design and strategy. Brown (2008) notes that design thinking is ‘‘a discipline that uses the designer’s sensibility and methods to match people’s needs with what is technically feasible and what business strategy can convert into customer value and market opportunities’’ (p. 1). The value that design thinking brings to an organization is a different way of framing situations and possibilities, doing things, and tackling problems: essentially a cultural transformation of the way it undertakes its business. The work of Martin (2009) has clearly shown the generalized differences between design thinking and business thinking, highlighting many instances in which these differences have been overcome, but also noting the many obstacles of trying to unify both approaches within an organization. Liedtka (2010) encourages firms to try and persist in overcoming these barriers, as she has noted that ‘‘business strategy desperately needs design ... because design is all about action and business strategy too often turns out to be only about talk ... fewer than 10 percent of new strategies are ever fully executed’’ (p. 9).

What information-related activities do people with ESKD use?

Background Information practice is an emerging area of research that seeks to reveal how people learn to connect with the complex multimodal information landscapes that informs their ability to make decisions. Previous research has identified that people with end stage kidney disease (ESKD) tend to adopt a ‘received’ or ‘engaged’ view of information but little is known about the activities of information practice. Objectives This research project sought to identify the: i) information-related activities; and ii) how information is used. Methods Using a constructivist qualitative methodology, ten people with ESKD living in a large metropolitan city were purposively selected and interviewed. Data was subject to thematic analysis by researchers from nursing and information science. Saturation of themes was achieved. Results Participants were between 38 and 72 years, had been receiving kidney replacement therapy from 2 weeks to 31 years. Eight participants reported having access to the internet but none participated in chat rooms. The activities were conceptualized into themes as listening, seeking, searching, sharing and observing. These activities enabled people to create, reflect on and evaluate the information needed to inform their decision-making Conclusion/Application to Clinical Practice The information practice research approach will enable a better understanding of the underlying relationship between information, knowledge and experience to be better understood. For renal nurses who are involved in patient education being able to recognise the way people use information will assist in individualizing educational sessions and tailoring teaching strategies to make it more meaningful.

Integrating robotics in primary school activities

This article outlines the integration of robotics in two settings in a primary school. This initiative was part of an Australian Research Council project which was undertaken at this school. The article highlights how robotics was integrated in a technology unit in a year four class. It also explains how it was embedded into an after-school program which catered for students from years five to seven. From these experiences further possibilities of engaging with robotics are also discussed.

The moderating effect of knowledge sharing on the relationship between manufacturing activities and business performance

This paper investigates the critical role of knowledge sharing (KS) in leveraging manufacturing activities, namely integrated supplier management (ISM) and new product development (NPD) to improve business performance (BP) within the context of Taiwanese electronic manufacturing companies. The research adopted a sequential mixed method research design, which provided both quantitative empirical evidence as well as qualitative insights, into the moderating effect of KS on the relationships between these two core manufacturing activities and BP. First, a questionnaire survey was administered, which resulted in a sample of 170 managerial and technical professionals providing their opinions on KS, NPD and ISM activities and the BP level within their respective companies. On the basis of the collected data, factor analysis was used to verify the measurement model, followed by correlation analysis to explore factor interrelationships, and finally moderated regression analyses to extract the moderating effects of KS on the relationships of NPD and ISM with BP. Following the quantitative study, six semi-structured interviews were conducted to provide qualitative in-depth insights into the value added from KS practices to the targeted manufacturing activities and the extent of its leveraging power. Results from quantitative statistical analysis indicated that KS, NPD and ISM all have a significant positive impact on BP. Specifically, IT infrastructure and open communication were identified as the two types of KS practices that could facilitate enriched supplier evaluation and selection, empower active employee involvement in the design process, and provide support for product simplification and the modular design process, thereby improving manufacturing performance and strengthening company competitiveness. The interviews authenticated many of the empirical findings, suggesting that in the contemporary manufacturing context KS has become an integral part of many ISM and NPD activities and when embedded properly can lead to an improvement in BP. The paper also highlights a number of useful implications for manufacturing companies seeking to leverage their BP through innovative and sustained KS practices.