Molecular and cellular aspects of copper transport in developing mammals

Copper is an essential trace element that requires tightly regulated homeostatic mechanisms to ensure adequate supplies without any toxic effects because of the ability of the metal ion to catalyze the formation of free radicals. The Cu-ATPases, ATP7A and ATP7B, play an important role in the physiological regulation of copper. Adequate supplies of copper are particularly important in developing animals, and in humans this is illustrated by mutations of ATP7A that cause the copper deficiency condition Menkes disease, which is fatal in early childhood. In contrast, mutations in ATP7B result in the genetic toxicosis, Wilson disease. We propose that the physiological regulation of copper is accomplished mainly by the intracellular copper-regulated trafficking of the Cu-ATPases. This process allows the overall copper status in the body to be maintained when levels of copper in the diet alter. A study of the defects in mouse models of Menkes and Wilson diseases has demonstrated that both ATPases play an important role in supplying copper to the developing fetus and neonate

Molecular and cellular characterisation of the zinc uptake (Znu) system of Nostoc punctiforme

Metal homoeostasis in cyanobacteria is based on uptake and export systems that are controlled by their own regulators. This study characterises the zinc uptake (Znu) system in Nostoc punctiforme. The system was found to comprise of three subunits in an ACB operon: a Zn2+-binding protein (ZnuA18), a transmembrane domain (ZnuB) and an ATPase (ZnuC). These proteins are encoded within the znu operon regulated by a zinc uptake transcription repressor (Zur). Interestingly, a second Zn2+-binding protein (ZnuA08) was also identified at a distal genomic location. Interactions between components of the ZnuACB system were investigated using knockouts of the individual genes. The znuA08-, znuA18-, znuB- and znuC- mutants displayed overall reduced znuACB transcript levels, suggesting that all system components are required for normal expression of znu genes. Zinc uptake assays in the Zn2+-binding protein mutant strains showed that the disruption of znuA18 had a greater negative effect on zinc uptake than disruption of znuA08. Complementation studies in Escherichia coli indicated that both znuA08 and znuA18 were able to restore zinc uptake in a znuA- mutant, with znuA18 permitting the highest zinc uptake rate. The N. punctiforme zur was also able to complement the E. coli zur- mutant.

Oxidative stress : emerging mitochondrial and cellular themes and variations in neuronal injury

Oxidative stress plays a central role in neuronal injury and cell death in acute and chronic pathological conditions. The cellular responses to oxidative stress embrace changes in mitochondria and other organelles, notably endoplasmic reticulum, and can lead to a number of cell death paradigms, which cover a spectrum from apoptosis to necrosis and include autophagy. In Alzheimer's disease, and other pathologies including Parkinson's disease, protein aggregation provides further cellular stresses that can initiate or feed into the pathways to cell death engendered by oxidative stress. Specific attention is paid here to mitochondrial dysfunction and programmed cell death, and the diverse modes of cell death mediated by mitochondria under oxidative stress. Novel insights into cellular responses to neuronal oxidative stress from a range of different stressors can be gained by detailed transcriptomics analyses. Such studies at the cellular level provide the key for understanding the molecular and cellular pathways whereby neurons respond to oxidative stress and undergo injury and death. These considerations underpin the development of detailed knowledge in more complex integrated systems, up to the intact human bearing the neuropathology, facilitating therapeutic advances.

At a glance: cellular biology for engineers

Engineering contributions have played an important role in the rise and evolution of cellular biology. Engineering technologies have helped biologists to explore the living organisms at cellular and molecular levels, and have created new opportunities to tackle the unsolved biological problems. There is now a growing demand to further expand the role of engineering in cellular biology research. For an engineer to play an effective role in cellular biology, the first essential step is to understand the cells and their components. However, the stumbling block of this step is to comprehend the information given in the cellular biology literature because it best suits the readers with a biological background. This paper aims to overcome this bottleneck by describing the human cell components as micro-plants that form cells as micro-bio-factories. This concept can accelerate the engineers’ comprehension of the subject. In this paper, first the structure and function of different cell components are described. In addition, the engineering attempts to mimic various cell components through numerical modelling or physical implementation are highlighted. Next, the interaction of different cell components that facilitate complicated chemical processes, such as energy generation and protein synthesis, are described. These complex interactions are translated into simple flow diagrams, generally used by engineers to represent multi-component processes.

Molecular and immunological analysis of hen's egg yolk allergens with a focus on YGP42 (Gal d 6)

Allergy to hen's (Gallus domesticus) egg white is one of the most common forms of food allergy. Allergy to hen's yolk also exists however, to a lesser extent when compared to egg white allergy. Two minor allergens from the hen's egg yolk known as α-livetin (Gal d 5) and YGP42 (Gal d 6) were discovered recently. In this study, we investigated whether sensitization to egg white is associated with reactivity to egg yolk as well. Sera obtained from 25 patients with allergy to egg white were tested for specific IgE binding for egg yolk proteins through western immunoblotting. 36% of patients were found with true IgE-sensitization against egg yolk proteins. It was found that most of the IgE reactive yolk proteins were fragments of major precursor proteins of hen; vitellogenin-1 (VTG-1), vitellogenin-2 (VTG-2) and apolipoprotein B (Apo B). The egg yolk allergen Gal d 6 is the C-terminal part of VTG-1 and was found to be allergenic in significant percentage of egg white allergy patients. These results highlight the significance of Gal d 6 as an important allergen of egg yolk. Therefore, the secondary aim of this study involved developing a recombinant version of YGP42 in an Escherichia coli expression system. Recombinant Gal d 6 (rGal d6) was expressed as a fusion peptide with a 6 × His tag and purified using metal chelating resin. The inhibition ELISA results showed that rYGP42 was IgE reactive and was able to inhibit IgE binding to crude egg yolk (CEY) by up to 30%. Traditionally, it was thought that allergy to egg yolk occurred independently from sensitization to egg white. This study underlies the importance of concomitant sensitization to egg yolk proteins in patients allergic to egg white. Evidence reported in this study strongly suggests that egg yolk has potentially undiscovered allergens and therefore warrants further investigation. Furthermore, IgE reactive Gal d 6 presented in this study has the potential to be used in diagnosis and immunotherapy to treat egg allergy.

Conservation ecology and breeding biology of the white-browed treecreeper, Climacteris affinis

The White-browed Treecreeper Climacteris affinis is one of many woodland-dependent birds that are at risk from the encroachment of human-dominated land-uses into natural landscapes. The White-browed Treecreeper inhabits semi-arid woodlands in north-west Victoria, Australia, a vegetation community that has undergone extreme modification in the last century due to the expansion of agriculture in the region. Extant woodlands represent only 10% of the original woodland cover in the region, and are highly fragmented and disturbed in many districts. Thus, the survival of the White-browed Treecreeper may depend on active management. However, current knowledge of the ecology and biology of this species is virtually non-existent, and inadequate for informed and effective conservation actions. The aim of this thesis is to redress this situation and provide the ecological basis for sound conservation management of the species. The thesis consists of two parts: an investigation of habitat use at three spatial scales and a study of the social organization, nesting requirements, breeding behaviour and reproductive success of a population of White-browed Treecreepers. Fifty-six patches of remnant woodland in north-west Victoria were surveyed to determine the factors affecting the occurrence of the White-browed Treecreeper at the regional scale. It was detected in 16 patches, and was largely confined to two core districts - Yarrara and, Wyperfeld (Pine Plains). The floristic composition of the dominant tree species was an important determinant of patch occupancy, with the results providing quantitative support for the previously suspected affinity for Belah Casuarina pauper and Slender Cypress-pine Callitris gracilis — Buloke Allocasuarina luehmannii woodlands. However, the absence of the White-browed Treecreeper from several districts was due to factors other than a lack of appropriate habitat. Demographic isolation - the distance from the focal patch to the nearest population of the White-browed Treecreeper - was the most important variable in explaining variation in patch occupancy. Patches isolated from other treecreeper populations by more than 8.3 km in landscapes of non-preferred native vegetation, and 3 km in agricultural landscapes, were unlikely to support the White-browed Treecreeper. The impact of habitat loss and fragmentation on the capacity of individuals to move through the landscape (i.e. functional connectivity) is considered in relation to disruption to dispersal and migration, and the potential collapse of local metapopulations. Habitat use was then examined in a network of patches and linear strips of Belah woodland embedded in a predominantly cultivated landscape. A minimum area of 18.5 ha of Belah woodland was identified as the most important criterion for patch occupancy at the local scale. This landscape appeared to be permeable to movement by the White-browed Treecreeper, facilitated by the extensive network of linear habitat, and clusters of small to medium fragments. The third scale of habitat use investigated the frequency of use of 1-ha plots within tracts of occupied woodland. It is important to discriminate between habitat traits that operate at the population level, and those that act as proximate cues for habitat selection by individuals. Woodlands that have high tree density, extensive cover of low-stature shrubs, abundant lichen, a complex vertical structure, and relatively low cover of grass and herbs are likely to support larger populations of the White-browed Treecreeper. However, individuals appeared to be using tree dominance (positive) and tall shrub cover (negative) as proximate environmental stimuli for habitat selectivity. A relatively high cover of ground lichen, which probably reflects a ground layer with low disturbance and high structural complexity, was also a reliable indicator of habitat use. Predictive models were developed which could be used to plan vegetation management to enhance habitat for the White-browed Treecreeper. The results of the regional, landscape and patch-scale investigations emphasise that factors operating at multiple spatial scales influence the suitability of remnant vegetation as habitat for the White-browed Treecreeper. The White-browed Treecreeper is typical of many small Australian passerines in that it has high annual survival, small clutches, a long breeding season, multiple broods and relatively low reproductive rates. Reproductive effort is adjusted through the number of clutches laid rather than clutch size. They occupy relatively large, all-purpose territories throughout the year. However, unlike many group territorial birds, territory size was not related to the number of occupants. The White-browed Treecreeper nests in tree hollows. They select hollows with a southerly orientation where possible, and prefer hollows that were higher from the ground. At Yarrara, there was considerable spatial variation in hollow abundance that, in concert with territorial constraints, restricted the actual availability of hollows to less than the absolute abundance of hollows. Thus, the availability of suitable hollows may limit reproductive productivity in some territories, although the magnitude of this constraint on overall population growth is predicted to be small. However, lack of recruitment of hollow-bearing trees would increase the potential for hollow availability to limit population growth. This prospect is particularly relevant in grazed remnants and those outside the reserve system. Facultative cooperative breeding was confirmed, with groups formed through male philopatry. Consequently, natal dispersal is female-biased, although there was no skew in the sex ratio of the fledglings or the general adult population. Helpers were observed performing all activities associated with parenting except copulation and brooding. Cooperatively breeding groups enjoyed higher fledgling productivity than simple pairs, after statistically accounting for territory and parental quality. However, the difference reflected increased productivity in the 1999-breeding season only, when climatic conditions were more favourable than in 1998. Breeding commenced earlier in 1999, and all breeding units were more likely to attempt a second brood. However, only breeders with helpers were successful in fledging second brood young, and it was this difference that accounted for the overall discrepancy in productivity. The key mechanism for increased success in cooperative groups was a reduction hi the interval between first and second broods, facilitated by compensatory reductions in the level of care to the first brood. Thus, females with helpers probably achieved significant energetic savings during this period, which enabled them to re-lay sooner. Furthermore, they were able to recommence nesting when the fledglings from the first brood were younger because there were more adults to feed the dependent juveniles. The current utility, and possible evolutionary pathways, of cooperative breeding is examined from the perspective of both breeders and helpers. Breeders benefit through enhanced fledgling productivity in good breeding conditions and a reduction in the burden of parental care, which may impart significant energetic savings. Further, breeders may facilitate philopatry as a means for ensuring a minimum level of reproductive success. Helpers benefit through an increase in their inclusive fitness in the absence of opportunities for independent breeding (i.e. ecological constraints) and access to breeding vacancies in the natal or adjacent territories (i.e. benefits of philopatry). However, the majority of breeding unit-years comprised unassisted breeders, which suggests that pairs are selectively favoured under certain environmental or demographic conditions.

Sindbis virus vectors elicit hemagglutinin-specific humoral and cellular immune responses and offer a dose-sparing strategy for vaccination

We report here on the use of a Sindbis virus-based DNA-launch RNA replicon vector (pSIN-HA) that expresses influenza hemagglutinin (HA) as an immunogen. Immunization of mice with pSIN-HA generated anti-HA antibody and CTL responses and resulted in lower lung viral titers after influenza challenge when compared to controls. Importantly, immunization with a low dose of pSIN-HA mediated significantly reduced lung viral titers following challenge at 43 weeks after the final immunization. In contrast, immunization with a non-replicon DNA vector expressing HA failed to mediate reduced lung viral titer at the same dose. This demonstrated the dose-sparing capacity of the SIN vector system and its ability to stimulate long-term memory responses, properties that are highly desirable in any vaccine formulation.

Liquids intermediate between 'molecular' and 'ionic' liquids : liquid ion pairs?

Ionic liquids comprised of tetradecyltrihexyl- and tetrabutyl- phosphonium cations paired with chloride or sulfonyl amide anions exhibit properties that reflect strong ion association, including comparatively low viscosity as well as a degree of volatility, and hence exemplify an interesting intermediate state between true ionic and true molecular liquids.