A role for the phagosome in cytokine secretion

Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor α (TNFα) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNFα and expands the membrane for phagocytosis.

Syntaxin 6 and Vti1b form a novel SNARE complex, which is upregulated in activated macrophages to facilitate exocytosis of TNF.

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNF; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNF� vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNF� trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

Association of immunoproteasomes with the endoplasmic reticulum

Proteasomes are complex multisubunit proteases which play a critical role in intracellular proteolysis. Immunoproteasomes, which contain three c-interferon-inducible subunits, are a subset of proteasomes which have a specialized function in antigen processing for presentation by the MHC class I pathway. Two of the c-interferon inducible subunits, LMP2 and LMP7, are encoded within the MHC class II region adjacent to the two TAP (transporter associated with antigen presentation) genes. We have investigated the localization of immunoproteasomes using monoclonal antibodies to LMP2 and LMP7. Immunoproteasomes were strongly enriched around the endoplasmic reticulum as judged by double-immuno¯uorescence experiments with anticalreticulin antibodies, but were also present in the nucleus and throughout the cytosol. In contrast, proteasome subunit C2, which is present in all proteasomes, was found to be evenly distributed throughout the cytoplasm and in the nucleus, as was the delta subunit, which is replaced by LMP2 in immunoproteasomes. c-Interferon increased the level of immunoproteasomes, but had no effect on their distribution. Our results provide the ®rst direct evidence that immunoproteasomes are strongly enriched at the endoplasmic reticulum, where they may be located close to the TAP transporter to provide efficient transport of peptides into the lumen of the endoplasmic recticulum for association with MHC class I molecules.

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable c-interferoninducible catalytic b-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit speci®c antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core a-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some signi®cant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immuno¯uorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following cinterferon treatment of cultured cells but c-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Phosphorylation of ATPase subunits of the 26S proteasome

Abstract The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2DPAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S oteasomes were immunoprecipitated as above and dissociated and Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.

Proteasome activities decrease during dexamethasone-induced apoptosis of thymocytes

The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially puri®ed from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-¯uoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-¯uoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in .itro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.

Evolution of the extinct Sabretooths and the American cheetah-like cat

The sabretooths (Smilodon and Homotherium) and the American cheetah-like cat (Miracinonyx) were the top predators in Late Pleistocene America, but became extinct about 13 thousand years ago. As the evolutionary history of these taxa remains poorly understood , we analysed their phylogenetic relationship to extant felids. In contrast to previous molecular studies , our results show that the sabretooths diverge early and are not closely related to any living cats. This supports their morphological placement in a separate subfamily (Machairodontinae). Despite its remarkable morphological similarity to the African cheetah (Acinonyx jubatus), Miracinonyx appears to have evolved from a puma-like ancestor, presumably in response to similar ecological pressures.

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

Regulation of proteasome structure and function

Proteasomes are cylindrical particles made up of a stack of four heptameric rings. In animal cells the outer rings are made up of 7 different types of alpha subunits and the inner rings are composed of 7 out of 10 possible different beta subunits. Regulatory complexes can bind to the ends of the cylinder.We have investigated aspects of the assembly, activity and subunit composition of core proteasome particles and 26S proteasomes, the localization of proteasome subpopulations, and the possible role of phosphorylation in determining proteasome localization, activities and association with regulatory components.

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the c~ and /3 subunits of the simpler proteasome isolated from Thermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that /3-type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these/3-type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulated in vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.

Resourcing Early Learners : New Networks, New Actors

The landscape of early childhood education and care is changing. Governments world-wide are assuming increasing authority in relation to child-rearing in the years before school entry, beyond the traditional role in assisting parents to do the best they can by their children. As part of a social agenda aimed at forming citizens well prepared to play an active part in a globalised knowledge economy, the idea of ‘early learning’ expresses the necessity of engaging caregivers right from the start of children’s lives. Nichols, Rowsell, Rainbird, and Nixon investigate this trend over three years, in two countries, and three contrasting regions, by setting themselves the task of tracing every service and agent offering resources under the banner of early learning. Far from a dry catalogue, the study involves in-depth ethnographic research in fascinating spaces such as a church-run centre for African refugee women and children, a state-of-the-art community library and an Australian country town. Included is an unprecedented inventory of an entire suburban mall. Richly visually documented, the study employs emerging methods such as Google-mapping to trace the travels of actual parents as they search for particular resources. Each chapter features a context investigated in this large, international study: the library, the mall, the clinic, and the church. The author team unravels new spaces and new networks at work in early childhood literacy and development.

Fundraising and philanthropy : engaging research and practice

The social and corporate trends over five years (1996 - 2000) in Australia clearly demonstrate the need for the nonprofit sector to engage in predictability forecasting to build viable philanthropic partnerships. As business and private enterprise practices have become more common in the management of fundraising effectiveness, nonprofits are in danger of reducing the value of their cause and likewise the cause or need of corporate and individual donors. Shortterm partnerships with short-term objectives do not achieve an outcome of sustainability. This paper analyses the theories of fundraising and philanthropy in the context of the changing Australian environment, and proposes a value measurement approach to the inputs and outputs of nonprofit organisations. By engaging in research, nonprofits are more likely to achieve productivity in fundraising and philanthropic practice.

Co-management to solve homelessness : wicked solutions to wicked problems

While governments are engaged in developing social policy responses to address wicked issues such as poverty, homelessness, drug addiction and crime, long term resolution of these issues through government policy making and state-based programmatic action has remained elusive. The use of vehicles for joint action and partnership between government and the community sector such as co-management has been offered as a way of harnessing productive capability and innovative capacity of both these sectors to resolve these complex problems. However, it is suggested that while there is a well advanced agenda with the intent for collaboration and partnership, working with the models for undertaking this joint action are not well understood and have not been fully developed or evaluated. This chapter examines new approaches to resolving the wicked issue of homelessness through applying the lens of co-management to understand the complexities of this issue and its resolution. The chapter analyses an attempt to move away from traditional bureaucratic structures of welfare departments, operating through single functional ‘silos’ to a new horizontal ‘hub-based’ model of service delivery that seeks to integrate actors across many different service areas and organizations. The chapter explores case studies of co-management in the establishment, development and operation of service hubs to address homelessness. We argue that the response to homelessness needs a ‘wicked solution’ that goes beyond simply providing shelter to those in need. The case of the hub models of community sector organizations working across organizational boundaries is evaluated to determine whether this approach can be considered successful co-managing of an innovative initiative, and understanding the requirements for developing, improving and extending this model. The role of the third sector in co-managing public services is examined through the in-depth case studies and the results are presented together with an assessment of how co-management can contribute to service quality and service management in public services.

Biodiversity–productivity relationships in small-scale mixed-species plantations using native species in Leyte province, Philippines

In this study, we investigate the relationship between tree species diversity and production in 18 mixed-species plantations established under the Rainforestation Farming system in Leyte province, the Philippines. The aim was to quantify productivity in the mixed-species plantations in comparison to the monocultures, and identify key drivers of productivity including environmental conditions, stand structural characteristics and surrogate measures of biodiversity, i.e. species richness, Shannon’s diversity index and functional groups. We found that monocultures had a much higher productivity than mixtures of the same and other species. In the mixtures, biodiversity and productivity did not have a simple relationship. Instead the proportion of exotic and native species, and the proportion of fast-growing species had a marginally significant positive effect on stand productivity, but no significant relationship was found with species richness or Shannon’s diversity. Instead stand structural characteristics such as density and age were the strongest drivers of increased productivity. Production levels within the mixed-species plantations varied significantly between sites. Overall, we found that the productivity of mixed species plantations was driven more by the characteristics of species present and stand structural characteristics then by simply the number and abundance of species, which suggests management practices are key for balancing multiple objectives to meet sustainable development needs.

Plastic traits of an exotic grass contribute to its abundance but are not always favourable

In herbaceous ecosystems worldwide, biodiversity has been negatively impacted by changed grazing regimes and nutrient enrichment. Altered disturbance regimes are thought to favour invasive species that have a high phenotypic plasticity, although most studies measure plasticity under controlled conditions in the greenhouse and then assume plasticity is an advantage in the field. Here, we compare trait plasticity between three co-occurring, C 4 perennial grass species, an invader Eragrostis curvula, and natives Eragrostis sororia and Aristida personata to grazing and fertilizer in a three-year field trial. We measured abundances and several leaf traits known to correlate with strategies used by plants to fix carbon and acquire resources, i.e. specific leaf area (SLA), leaf dry matter content (LDMC), leaf nutrient concentrations (N, C:N, P), assimilation rates (Amax) and photosynthetic nitrogen use efficiency (PNUE). In the control treatment (grazed only), trait values for SLA, leaf C:N ratios, Amax and PNUE differed significantly between the three grass species. When trait values were compared across treatments, E. curvula showed higher trait plasticity than the native grasses, and this correlated with an increase in abundance across all but the grazed/fertilized treatment. The native grasses showed little trait plasticity in response to the treatments. Aristida personata decreased significantly in the treatments where E. curvula increased, and E. sororia abundance increased possibly due to increased rainfall and not in response to treatments or invader abundance. Overall, we found that plasticity did not favour an increase in abundance of E. curvula under the grazed/fertilized treatment likely because leaf nutrient contents increased and subsequently its' palatability to consumers. E. curvula also displayed a higher resource use efficiency than the native grasses. These findings suggest resource conditions and disturbance regimes can be manipulated to disadvantage the success of even plastic exotic species.