Development of a fluorescence polarization binding assay for asialoglycoprotein receptor

Asialoglycoprotein receptor (ASGP-R) has been actively investigated for targeted delivery of therapeutic agents into hepatocytes because this receptor is selectively and highly expressed in liver and has a high internalization rate. Synthetic cluster glycopeptides (e.g., triGalNAc) bind with high affinity to ASGP-R and, when conjugated to a therapeutic agent, can drive receptor-mediated uptake in liver. We developed a novel fluorescent polarization (FP) ASGP-R binding assay to determine the binding affinities of ASGP-R-targeted molecules. The assay was performed in 96-well microplates using membrane preparations from rat liver as a source of ASGP-R and Cy5 fluorophore-labeled triGalNAc synthetic ligand as a tracer. This high-throughput homogeneous assay demonstrates advantages over existing multistep methods in that it minimizes both time and resources spent in determining binding affinities to ASGP-R. At the optimized conditions, a Z' factor of 0.73 was achieved in a 96-well format.     

Host behaviour and physiology underpin individual variation in avian influenza virus infection in migratory Bewick's swans

Individual variation in infection modulates both the dynamics of pathogens and their impact on host populations. It is therefore crucial to identify differential patterns of infection and understand the mechanisms responsible. Yet our understanding of infection heterogeneity in wildlife is limited, even for important zoonotic host-pathogen systems, owing to the intractability of host status prior to infection. Using novel applications of stable isotope ecology and eco-immunology, we distinguish antecedent behavioural and physiological traits associated with avian influenza virus (AIV) infection in free-living Bewick's swans (Cygnus columbianus bewickii). Swans infected with AIV exhibited higher serum δ13C (-25.3±0.4) than their non-infected counterparts (-26.3±0.2). Thus, individuals preferentially foraging in aquatic rather than terrestrial habitats experienced a higher risk of infection, suggesting that the abiotic requirements of AIV give rise to heterogeneity in pathogen exposure. Juveniles were more likely to be infected (30.8% compared with 11.3% for adults), shed approximately 15-fold higher quantity of virus and exhibited a lower specific immune response than adults. Together, these results demonstrate the potential for heterogeneity in infection to have a profound influence on the dynamics of pathogens, with concomitant impacts on host habitat selection and fitness.     

Blunted neuronal calcium response to hypoxia in naked mole-rat hippocampus

Naked mole-rats are highly social and strictly subterranean rodents that live in large communal colonies in sealed and chronically oxygen-depleted burrows. Brain slices from naked mole-rats show extreme tolerance to hypoxia compared to slices from other mammals, as indicated by maintenance of synaptic transmission under more hypoxic conditions and three fold longer latency to anoxic depolarization. A key factor in determining whether or not the cellular response to hypoxia is reversible or leads to cell death may be the elevation of intracellular calcium concentration. In the present study, we used fluorescent imaging techniques to measure relative intracellular calcium changes in CA1 pyramidal cells of hippocampal slices during hypoxia. We found that calcium accumulation during hypoxia was significantly and substantially attenuated in slices from naked mole-rats compared to slices from laboratory mice. This was the case for both neonatal (postnatal day 6) and older (postnatal day 20) age groups. Furthermore, while both species demonstrated more calcium accumulation at older ages, the older naked mole-rats showed a smaller calcium accumulation response than even the younger mice. A blunted intracellular calcium response to hypoxia may contribute to the extreme hypoxia tolerance of naked mole-rat neurons. The results are discussed in terms of a general hypothesis that a very prolonged or arrested developmental process may allow adult naked mole-rat brain to retain the hypoxia tolerance normally only seen in neonatal mammals.     

Heritability and phenotypic variation of canine hip dysplasia radiographic traits in a cohort of Australian German shepherd dogs

Canine Hip Dysplasia (CHD) is a common, painful and debilitating orthopaedic disorder of dogs with a partly genetic, multifactorial aetiology. Worldwide, potential breeding dogs are evaluated for CHD using radiographically based screening schemes such as the nine ordinally-scored British Veterinary Association Hip Traits (BVAHTs). The effectiveness of selective breeding based on screening results requires that a significant proportion of the phenotypic variation is caused by the presence of favourable alleles segregating in the population. This proportion, heritability, was measured in a cohort of 13,124 Australian German Shepherd Dogs born between 1976 and 2005, displaying phenotypic variation for BVAHTs, using ordinal, linear and binary mixed models fitted by a Restricted Maximum Likelihood method. Heritability estimates for the nine BVAHTs ranged from 0.14-0.24 (ordinal models), 0.14-0.25 (linear models) and 0.12-0.40 (binary models). Heritability for the summed BVAHT phenotype was 0.30 ± 0.02. The presence of heritable variation demonstrates that selection based on BVAHTs has the potential to improve BVAHT scores in the population. Assuming a genetic correlation between BVAHT scores and CHD-related pain and dysfunction, the welfare of Australian German Shepherds can be improved by continuing to consider BVAHT scores in the selection of breeding dogs, but that as heritability values are only moderate in magnitude the accuracy, and effectiveness, of selection could be improved by the use of Estimated Breeding Values in preference to solely phenotype based selection of breeding animals.     

Carbon catabolite repression in <Emphasis Type="Italic">Thermoanaerobacterium saccharolyticum</Emphasis>

Background

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.

Results

We report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.

Conclusion

Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

     

Leaf litter input mediates tadpole performance across forest canopy treatments

Understanding the mechanisms limiting the distributions of organisms is necessary for predicting changes in community composition along habitat gradients. In many areas of the USA, land originally cleared for agriculture has been undergoing a process of reforestation, creating a gradient of canopy cover. For small temporary wetlands, this gradient can alter abiotic conditions and influence the resource base of wetland food webs by affecting litter inputs. As distributions of amphibians and many other temporary wetland taxa correlate with canopy cover, we experimentally manipulated shade levels and litter types in pond mesocosms to explore mechanisms limiting species performance in wetlands with canopy cover. Most differences between ponds were mediated by litter type rather than direct effects of shading. Although all three amphibian species tested are open-canopy specialists, spring peepers were the only species to show decreased survival in shaded ponds. Pond litter type generally had strong effects on growth and development rates, with tadpoles of two species in grass litter ponds growing to twice the size of, and metamorphosing 7 days earlier than, those in leaf litter ponds. Contrary to our initial hypothesis, shade level and litter type showed very few significant interactions. Our results indicate that the effects of shading cannot be considered in isolation of vegetation changes in pond basins when evaluating the effects of forest succession on temporary pond communities.     

Yeast Est2p affects telomere length by influencing association of Rap1p with telomeric chromatin

In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.     

Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

During maturation, chondrocytes undergo changes in morphology, matrix production, and gene expression; however, it remains unclear whether these are interrelated. In this study, we examined whether Rho GTPases were involved in these regulatory interplays. Levels of active Rho GTPases were assayed in immature and mature primary chondrocytes. We found that activation of Rac-1 and Cdc42 increased with maturation, whereas RhoA levels remained unchanged. GFP-tagged Rho GTPases tracked cellular localization. Rac-1 was enriched at the cell membrane where it co-localized with cortical actin, while RhoA and Cdc42 were cytoplasmic. To test the roles of Rac-1 in chondrocyte maturation, we force-expressed constitutively active or dominant negative forms of Rac-1 and assessed phenotypic consequences in primary chondrocytes. Activated Rac-1 expression induced chondrocyte enlargement and increased matrix metalloproteinase expression, which are characteristic of mature chondrocytes. Conversely, Rac-1 inactivation diminished adhesion, decreased alkaline phosphatase activity, and stimulated functions typical of immature chondrocytes. Exposure to a pro-maturation factor, Wnt3A, induced a flattened and enlarged morphology accompanied by peripheral Rac-1 re-arrangement. Wnt3A stimulated Tiam1 expression and Rac-1 activation, while DN-Rac-1 inhibited Wnt3A-induced cell spreading. Our data provide strong evidence that Rac-1 coordinates changes in chondrocyte phenotype and function and stimulates the maturation process essential for skeletal development.     

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.