Green fluorescent protein as a tool to study Glut4 trafficking

Glut4 is the principal glucose transporter expressed in the peripheral tissues. In response to insulin, Glut4 moves from an intracellular compartment to the plasma membrane in a process called translocation. Once at the cell surface, Glut4 transports glucose across the plasma membrane into the cell and thus regulates whole body glucose homeostasis. At the molecular level we tagged Glut4 with Green Fluorescent Protein (GFP) in order to glean further insight into the translocation process. The native fluorescence of GFP then allowed us to analyse the trafficking of GFP-tagged Glut4 chimeras in real time using confocal microscopy. Glut4-GFP expressed in 3T3-L1 fibroblasts or fully differentiated adipocytes, displayed an intracellular distribution that only partially overlapped with the early endosomal compartment. However, Glut4-GFP constructs that carried mutations in N- and C- terminus targeting motifs were localised to the plasma membrane. Time lapse confocal analysis revealed the dynamic nature of Glut4-GFP-containing vesicles in the basal state. When Glut4~GFP-expressing adipocytes were treated with insulin, Glut4-GFP translocated to the plasma membrane. Translocation was rapid, analogous to endogenous Glut4 translocation in 3T3-L1 adipocytes. However, upon insulin removal, Glut4-GFP failed to reinternalise. We suggest that this failure to reinternalise, while recycling normally in the basal state, reflects a Glut4-specific reinternalisation mechanism following insulin-stimulated translocation. Glut4-GFP exhibited a similar intracellular distribution when expressed in L6 muscle cells. Nitric oxide (NO) donors stimulate glucose uptake into L6 muscle cells. Using inhibitors of the nitric-oxide signalling pathway and Glut4-GFP we investigated the mechanism by which NO-donors increase glucose transport into L6 cells. Recent research has also implicated ADP- Ribosylation Factors (ARFs) in insulin-stimulated Glut4 translocation. ARFs are small molecular weight GTPases. By site-directed mutagenesis we endeavoured to change the specificity of the ARFS and ARF6 isoforms from GTP to XTP. These mutations would allow us to control ARF5/6 function in stable 3T3-L1 cell lines. Our goal is then to analyse the contribution of ARFS and ARF6 to insulin stimulated Glut4-GFP translocation

The 6dF Galaxy Survey: Dependence of halo occupation on stellar mass

In this paper we study the stellar-mass dependence of galaxy clustering in the 6dF Galaxy Survey. The near-infrared selection of 6dFGS allows more reliable stellar mass estimates compared to optical bands used in other galaxy surveys. Using the Halo Occupation Distribution (HOD) model, we investigate the trend of dark matter halo mass and satellite fraction with stellar mass by measuring the projected correlation function, $w_p(r_p)$. We find that the typical halo mass ($M_1$) as well as the satellite power law index ($\alpha$) increase with stellar mass. This indicates, (1) that galaxies with higher stellar mass sit in more massive dark matter halos and (2) that these more massive dark matter halos accumulate satellites faster with growing mass compared to halos occupied by low stellar mass galaxies. Furthermore we find a relation between $M_1$ and the minimum dark matter halo mass ($M_{\rm min}$) of $M_1 \approx 22\,M_{\rm min}$, in agreement with similar findings for SDSS galaxies. The satellite fraction of 6dFGS galaxies declines with increasing stellar mass from 21% at $M_{\rm stellar} = 2.6\times10^{10}h^{-2}\,M_{\odot}$ to 12% at $M_{\rm stellar} = 5.4\times10^{10}h^{-2}\,M_{\odot}$ indicating that high stellar mass galaxies are more likely to be central galaxies. We compare our results to two different semi-analytic models derived from the Millennium Simulation, finding some disagreement. Our results can be used for placing new constraints on semi-analytic models in the future, particularly the behaviour of luminous red satellites. Finally we compare our results to studies of halo occupation using galaxy-galaxy weak lensing. We find good overall agreement, representing a valuable crosscheck for these two different tools of studying the matter distribution in the Universe.Comment: 17 pages, 11 figures. arXiv admin note: text overlap with arXiv:1104.2447 by other author

One pot ‘click’ reactions: tandem enantioselective biocatalytic epoxide ring opening and [3+2] azide alkyne cycloaddition

Halohydrin dehalogenase (HheC) can perform enantioselective azidolysis of aromatic epoxides to 1,2-azido alcohols which are subsequently ligated to alkynes producing chiral hydroxy triazoles in a one-pot procedure with excellent enantiomeric excess.

Interaction between temperature and sublethal infection with the amphibian chytrid fungus impacts a susceptible frog species

The amphibian chytrid fungus Batrachochytrium dendrobatidis is an emerging infectious pathogen present on every continent except Antarctica. It causes the disease chytridiomycosis in a subset of species but does not always result in disease or death for every host. Ambient temperature influences both amphibian metabolism and chytrid pathogenicity, however the interactive effects on host physiology is not well understood. We investigated the sublethal effect of B. dendrobatidis infection on a susceptible host, Litoria aurea to test (1) whether the infection load, metabolic activity, body fat and gonad size differed in L. aurea at either 24 degrees C or 12 degrees C ambient temperatures and (2) whether previous Bd infection caused long-term changes to body fat and gonad size. Litoria aurea in 12 degrees C treatments had higher infection loads of B. dendrobatidis and lower survivorship. Metabolic rate was higher and fat mass was lower in infected individuals and in animals in 24 degrees C treatments. Male L. aurea previously infected with B. dendrobatidis had smaller testes 5 months-post clearance of infection, an effect likely to translate to fitness costs in wild populations. These experiments demonstrate a physiological cost to sublethal B. dendrobatidis infection, which suggests a reduction in host fitness mediated by temperature in the host's environment regardless of whether infection leads to mortality

The Tiling Algorithm for the 6dF Galaxy Survey

The Six Degree Field Galaxy Survey (6dFGS) is a spectroscopic survey of the southern sky, which aims to provide positions and velocities of galaxies in the nearby Universe. We present here the adaptive tiling algorithm developed to place 6dFGS fields on the sky, and allocate targets to those fields. Optimal solutions to survey field placement are generally extremely difficult to find, especially in this era of large-scale galaxy surveys, as the space of available solutions is vast (2N dimensional) and false optimal solutions abound. The 6dFGS algorithm utilises the Metropolis (simulated annealing) method to overcome this problem. By design the algorithm gives uniform completeness independent of local density, so as to result in a highly complete and uniform observed sample. The adaptive tiling achieves a sampling rate of approximately 95%, a variation in the sampling uniformity of less than 5%, and an efficiency in terms of used fibres per field of greater than 90%. We have tested whether the tiling algorithm systematically biases the large-scale structure in the survey by studying the two-point correlation function of mock 6dF volumes. Our analysis shows that the constraints on fibre proximity with 6dF lead to under-estimating galaxy clustering on small scales (< 1 Mpc) by up to ~20%, but that the tiling introduces no significant sampling bias at larger scales.Comment: 11 pages, 7 figures. Full resolution version of the paper available from http://www.mso.anu.edu.au/6dFGS/ . Abridged version of abstract belo

The 6df galaxy survey: The near-infrared fundamental plane of early-type galaxies

We determine the near-infrared Fundamental Plane (FP) for ~10 4 early-type galaxies in the 6-degree Field Galaxy Survey (6dFGS). We fit the distribution of central velocity dispersion, near-infrared surface brightness and half-light radius with a 3D Gaussian model using a maximum-likelihood method. The model provides an excellent empirical fit to the observed FP distribution and the method proves robust and unbiased. Tests using simulations show that it gives superior results to regression techniques in the presence of significant and correlated uncertainties in all three parameters, censoring of the data by various selection effects and outliers in the data sample. For the 6dFGS J-band sample we find an FP with Re∝σ01.52±0.03Ie-0.89±0.01, similar to previous near-infrared determinations and consistent with the H- and K-band FPs once allowance is made for differences in mean colour. The overall scatter in R e about the FP is σ r = 29 per cent, and is the quadrature sum of an 18 per cent scatter due to observational errors and a 23 per cent intrinsic scatter. Because of the Gaussian distribution of galaxies in FP space, σ r is not the distance error, which we find to be σ d = 23 per cent. Using group richness and local density as measures of environment, and morphologies based on visual classifications, we find that the FP slopes do not vary with environment or morphology. However, for fixed velocity dispersion and surface brightness, field galaxies are on average 5 per cent larger than galaxies in groups or higher density environments, and the bulges of early-type spirals are on average 10 per cent larger than ellipticals and lenticulars. The residuals about the FP show significant trends with environment, morphology and stellar population. The strongest trend is with age, and we speculate that age is the most important systematic source of offsets from the FP, and may drive the other trends through its correlations with environment, morphology and metallicity. These results will inform our use of the near-infrared FP in deriving relative distances and peculiar velocities for 6dFGS galaxies

Post-fire recovery of woody plants in the New England Tableland Bioregion

The resprouting response of plant species to fire is a key life history trait that has profound effects on post-fire population dynamics and community composition. This study documents the post-fire response (resprouting and maturation times) of woody species in six contrasting formations in the New England Tableland Bioregion of eastern Australia. Rainforest had the highest proportion of resprouting woody taxa and rocky outcrops had the lowest. Surprisingly, no significant difference in the median maturation length was found among habitats, but the communities varied in the range of maturation times. Within these communities, seedlings of species killed by fire, mature faster than seedlings of species that resprout. The slowest maturing species were those that have canopy held seed banks and were killed by fire, and these were used as indicator species to examine fire immaturity risk. Finally, we examine whether current fire management immaturity thresholds appear to be appropriate for these communities and find they need to be amended