Fluorescence resonance energy transfer study of the associative state of membrane-bound complexes of complement proteins C5b-8

Human complement protein C8 was labeled with the fluorescent chromophores fluorescein-5-isothiocyanate (FITC), 3-(4-isothiocyanatophenyl)-7-diethylamine-4-methyl coumarin (IPM), eosin-5-isothiocyanate (EOS), or Texas Red (sulforhodamine-101-sulfonyl chloride; TR) with only minor reduction in the specific hemolytic activity of the protein. The distribution of C5b-8 complexes bound to sheep erythrocyte membranes was investigated by monitoring fluorescence resonance energy transfer (RET) between the following RET donor/acceptor pairs of labeled C8: FITC-C8/EOS-C8, IPM-C8/EOS-C8, and FITC-C8/TR-C8. On binding to membranes containing pre-formed C5b67 complexes, specific RET was detected for each of the donor/acceptor pairs of labeled C8 investigated. In contrast, no energy transfer was observed for these RET donor/acceptor pairs of labeled C8 incubated in the presence of control membranes or in membrane-free solution. On the basis of a consideration of the transfer efficiency that would be expected for donor/acceptor pairs of labeled C8 that were uniformly dispersed on the membrane surface, these results suggest that C5b-8 complexes are aggregated into polymeric clusters when membrane-bound. The efficiency of donor-C8 to acceptor-C8 RET--and the hemolytic activity of membrane-bound C5b-8 (in the absence of C9)--are both related to the surface density of membrane-bound C5b67, suggesting that the physical clustering of the membrane-inserted C5b-8 complex may be related to the expression of its cytolytic activity.     

Rethinking transnationalism through the second generation

The concept of transnationalism was developed to describe the ties maintained by migrants with their homelands and, until recently, little attention was given to the question of whether these ties would be continued by the migrants’ children, the ‘second generation’. This article discusses the transnational practices of second‐generation Tongans in Australia, using the research findings to reconsider some of the existing assumptions about what constitutes transnationalism. To fully understand second‐generation transnationalism, three forms of transnationalism are described: intradiasporic, indirect and forced transnationalism. Taking these forms into account reveals a more complex picture of the transnational ties maintained by migrants and their children than has been drawn in previous accounts.     

Ultrastructure of Macadamia (Proteaceae) embryos: implications for their breakage properties

BACKGROUND AND AIMS: Macadamia integrifolia, M. tetraphylla and their hybrids are cultivated for their edible kernels. Whole kernels, i.e. intact mature embryos with cotyledons fused together, are highly valued and breakage of embryos into halves results in loss of value for the commercial macadamia industry. The morphology and ultrastructure of the mature macadamia embryo, with particular emphasis on the break zone between cotyledons, were investigated. Differences in breakage between different macadamia cultivars were also examined. METHODS: Manual cracking was used to compare breakage in five cultivars and the ultrastructure of the break zone between the cotyledons was examined using light and transmission electron microscopy. KEY RESULTS: Breakage of macadamia embryos was strongly dependent on genotype of the female parent, with cultivars 'HAES 344' and 'HAES 741' much more likely to break than 'HV A16' and 'HAES 835'. Cotyledons were surrounded by a layer of cuticle resulting in a double cuticle in the break zone between the cotyledons. Three major differences have been found in the ultrastructure of the double cuticle between cultivars: a thicker cuticle in the low-whole cultivar; convolutions in the cuticle of a low-whole cultivar, and the presence of more electron-dense objects in the high-whole cultivar. CONCLUSIONS: Breakage of macadamia embryos depends on the cultivar, with clear ultrastructural differences in the break zone between cultivars. To ensure commercial benefits, macadamia breeding programs should identify germplasm with structural characteristics that ensure high percentages of whole kernel.     

Deep-diving behaviour of a whale shark Rhincodon typus during long-distance movement in the western Indian Ocean

A whale shark Rhincodon typus satellite tagged off the coast of Mozambique showed a highly directional movement across the Mozambique Channel and around the southern tip of Madagascar, a minimum distance of 1200 km in 87 days. Dives to depths well into the mesopelagic and bathypelagic zones (1286 m maximum depth) were recorded in a bathymetrically non-constraining habitat. The water temperature range recorded during the fish's movement was 3·4–29·9° C.     

The design and discovery of novel amide CCR5 antagonists

The synthesis of a range of novel amine-containing structures and their primary potency as inhibitors of HIV-1 fusion via blocking of the CCR5 receptor is described. The development of the medicinal chemistry strategy and SAR’s which led to the identification of the piperidine amide compounds 33 and 36 as excellent leads for further evaluation is described, along with key physicochemical data which highlighted their lead potential.     

Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1)

PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix alpha5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b.     

Regulation to Create Environments Conducive to Physical Activity: Understanding the Barriers and Facilitators at the Australian State Government Level

Introduction

Policy and regulatory interventions aimed at creating environments more conducive to physical activity (PA) are an important component of strategies to improve population levels of PA. However, many potentially effective policies are not being broadly implemented. This study sought to identify potential policy/regulatory interventions targeting PA environments, and barriers/facilitators to their implementation at the Australian state/territory government level.

Methods

In-depth interviews were conducted with senior representatives from state/territory governments, statutory authorities and non-government organisations (n = 40) to examine participants'': 1) suggestions for regulatory interventions to create environments more conducive to PA; 2) support for preselected regulatory interventions derived from a literature review. Thematic and constant comparative analyses were conducted.

Results

Policy interventions most commonly suggested by participants fell into two areas: 1) urban planning and provision of infrastructure to promote active travel; 2) discouraging the use of private motorised vehicles. Of the eleven preselected interventions presented to participants, interventions relating to walkability/cycling and PA facilities received greatest support. Interventions involving subsidisation (of public transport, PA-equipment) and the provision of more public transport infrastructure received least support. These were perceived as not economically viable or unlikely to increase PA levels. Dominant barriers were: the powerful ‘road lobby’, weaknesses in the planning system and the cost of potential interventions. Facilitators were: the provision of evidence, collaboration across sectors, and synergies with climate change/environment agendas.

Conclusion

This study points to how difficult it will be to achieve policy change when there is a powerful ‘road lobby’ and government investment prioritises road infrastructure over PA-promoting infrastructure. It highlights the pivotal role of the planning and transport sectors in implementing PA-promoting policy, however suggests the need for clearer guidelines and responsibilities for state and local government levels in these areas. Health outcomes need to be given more direct consideration and greater priority within non-health sectors.     

Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection

The Bacillus Calmette - Guerin (BCG) vaccine provides a critical but limited defense against Mycobacterium tuberculosis (M.tb). More than 60 years after the widespread introduction of BCG, there is an urgent need for a better vaccine. A large body of pre-clinical research continues to support ongoing clinical trials to assess whether viral vectors expressing M.tb antigens that are shared by BCG and M.tb, can be used alongside BCG to enhance protection. A major focus involves using multiple unique viral vectors to limit anti-vector immunity and thereby enhance responses to the insert antigen delivered. The successful introduction of viral vector vaccines to target M.tb and other pathogens will be reliant on reducing the costs when using multiple vectors and inhibiting the development of unwanted anti-vector responses that interfere with the response to insert antigen. This study examines methods to reduce the logistical costs of vaccination by mixing different viral vectors that share the same insert antigen in one vaccine; and whether combining different viral vectors reduces anti-vector immunity to improve immunogenicity to the insert antigen. Here we show that a homologous prime-boost regimen with a mixture of MVA (Modified Vaccinia virus Ankara) and Ad5 (human adenovirus type 5) vectors both expressing Ag85A in a single vaccine preparation is able to reduce anti-vector immunity, compared with a homologous prime-boost regimen with either vector alone. However, the level of immunogenicity induced by the homologous mixture remained comparable to that induced with single viral vectors and was less immunogenic than a heterologous Ad5 prime-MVA-boost regimen. These findings advance the understanding of how anti-vector immunity maybe reduced in viral vector vaccination regimens. Furthermore, an insight is provided to the impact on vaccine immunogenicity from altering vaccination methods to reduce the logistical demands of using separate vaccine preparations in the field.