Seasonal, tissue-specific regulation of Akt/protein kinase B and glycogen synthase in hibernators

Yellow-bellied marmots (Marmota flaviventris) exhibit a circannual cycle of hyperphagia and nutrient storage in the summer followed by hibernation in the winter. This annual cycle of body mass gain and loss is primarily due to large-scale accumulation of lipid in the summer, which is then mobilized and oxidized for energy during winter. The rapid and predictable change in body mass makes these animals ideal for studies investigating the molecular basis for body weight regulation. In the study described herein, we monitored seasonal changes in the protein levels and activity of a central regulator of anabolic metabolism, the serine-threonine kinase Akt-protein kinase B (Akt/PKB), during the months accompanying maximal weight gain and entry into hibernation (June-November). Interestingly, under fasting conditions, Akt/PKB demonstrated a tissue-specific seasonal activation. Specifically, although Akt/PKB levels did not change, the activity of Akt/PKB (isoforms 1/alpha and 2/beta) in white adipose tissue (WAT) increased significantly in July. Moreover, glycogen synthase, which lies downstream of Akt/PKB on a linear pathway linking the enzyme to the stimulation of glycogen synthesis, demonstrated a similar pattern of seasonal activation. By contrast, Akt/PKB activity in skeletal muscle peaked much later (i.e., September). These data suggest the existence of a novel, tissue-specific mechanism regulating Akt/PKB activation during periods of marked anabolism.     

Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation

The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.     

Nodamura virus RNA replication in Saccharomyces cerevisiae: heterologous gene expression allows replication-dependent colony formation

Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 10(4)-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.     

Fluorescent metal ion indicators based on benzoannelated crown systems: a green fluorescent indicator for intracellular sodium ions

The synthesis and metal binding properties of cation-sensitive fluorescent indicators intended for biological applications are described. The increase of the crown ether ring size enhances the affinity for larger cations, but weakens the fluorescent response and selectivity. A compound having a 15-crown-5 chelator directly attached to a 2,7-difluoroxanthenone fluorophore loads into live cells and responds to sodium ion concentration changes with large fluorescence increases in the visible wavelength range.     

Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-alpha activation

While cellular responses to low oxygen (O(2)) or hypoxia have been studied extensively, the precise identity of mammalian cellular O(2) sensors remains controversial. Using murine embryonic cells lacking cytochrome c, and therefore mitochondrial activity, we show that mitochondrial reactive oxygen species (mtROS) are essential for proper O(2) sensing and subsequent HIF-1 alpha and HIF-2 alpha stabilization at 1.5% O(2). In the absence of this signal, HIF-alpha subunits continue to be degraded. Furthermore, exogenous treatment with H(2)O(2) or severe O(2) deprivation is sufficient to stabilize HIF-alpha even in the absence of cytochrome c and functional mitochondria. These results provide genetic evidence indicating that mtROS act upstream of prolyl hydroxylases in regulating HIF-1 alpha and HIF-2 alpha in this O(2)-sensing pathway.     

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide metabolism of anaerobic hyperthermophiles

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+-->2CoA-SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.     

Spatial and genetic structure of directly-transmitted parasites reflects the distribution of their specific amphibian hosts

Parasite distributions depend on the local environment in which host infection occurs, and the surrounding landscape over which hosts move and transport their parasites. Although host and landscape effects on parasite prevalence and spatial distribution are difficult to observe directly, estimation of such relationships is necessary for understanding the spread of infections and parasite–habitat associations. Although parasite distributions are necessarily nested within host distributions, direct environmental influences on local infection or parasite effects on host dispersal could lead to distinct landscape or habitat relationships relative to their hosts. Our aim was to determine parasite spatial structure across a contiguous prairie by statistical modeling of parasite–landscape relationships combined with analysis of population genetic structure. We sampled northern leopard frogs (Lithobates pipiens) and wood frogs (L. sylvaticus) for host-specific lung nematodes (Rhabdias ranae and R. bakeri; respectively) across the Sheyenne National Grassland in southeastern North Dakota and developed primers for 13 microsatellite loci for Rhabdias. The two Rhabdias species exhibited different correlations with landscape characteristics that conformed with that of their hosts, indicating transmission is driven by host ecology, probably density, and not directly by the environment. There was evidence for localized, patchy spatial genetic structure, but no broader-scale geographic patterns, indicating no barriers to host and parasite dispersal. Nematodes cohabitating in an individual frog were most genetically similar. Worms within the same wetland were also genetically similar, indicating localized transmission and resulting wetland-scale patchiness are not completely obscured by broad-scale host–parasite dispersal. Beyond individual wetlands, we found no evidence of genetic isolation-by-distance or patchiness at the landscape-scale.     

Spring photosynthetic onset and net CO2 uptake in Alaska triggered by landscape thawing

The springtime transition to regional‐scale onset of photosynthesis and net ecosystem carbon uptake in boreal and tundra ecosystems are linked to the soil freeze–thaw state. We present evidence from diagnostic and inversion models constrained by satellite fluorescence and airborne CO₂ from 2012 to 2014 indicating the timing and magnitude of spring carbon uptake in Alaska correlates with landscape thaw and ecoregion. Landscape thaw in boreal forests typically occurs in late April (DOY 111 ± 7) with a 29 ± 6 day lag until photosynthetic onset. North Slope tundra thaws 3 weeks later (DOY 133 ± 5) but experiences only a 20 ± 5 day lag until photosynthetic onset. These time lag differences reflect efficient cold season adaptation in tundra shrub and the longer dehardening period for boreal evergreens. Despite the short transition from thaw to photosynthetic onset in tundra, synchrony of tundra respiration with snow melt and landscape thaw delays the transition from net carbon loss (at photosynthetic onset) to net uptake by 13 ± 7 days, thus reducing the tundra net carbon uptake period. Two global CO₂ inversions using a CASA‐GFED model prior estimate earlier northern high latitude net carbon uptake compared to our regional inversion, which we attribute to (i) early photosynthetic‐onset model prior bias, (ii) inverse method (scaling factor + optimization window), and (iii) sparsity of available Alaskan CO₂ observations. Another global inversion with zero prior estimates the same timing for net carbon uptake as the regional model but smaller seasonal amplitude. The analysis of Alaskan eddy covariance observations confirms regional scale findings for tundra, but indicates that photosynthesis and net carbon uptake occur up to 1 month earlier in evergreens than captured by models or CO₂ inversions, with better correlation to above‐freezing air temperature than date of primary thaw. Further collection and analysis of boreal evergreen species over multiple years and at additional subarctic flux towers are critically needed.     

Navigating north: how body mass and winds shape avian flight behaviours across a North American migratory flyway

The migratory patterns of birds have been the focus of ecologists for millennia. What behavioural traits underlie these remarkably consistent movements? Addressing this question is central to advancing our understanding of migratory flight strategies and requires the integration of information across levels of biological organisation, e.g. species to communities. Here, we combine species‐specific observations from the eBird citizen‐science database with observations aggregated from weather surveillance radars during spring migration in central North America. Our results confirm a core prediction of migration theory at an unprecedented national scale: body mass predicts variation in flight strategies across latitudes, with larger‐bodied species flying faster and compensating more for wind drift. We also find evidence that migrants travelling northward earlier in the spring increasingly compensate for wind drift at higher latitudes. This integration of information across biological scales provides new insight into patterns and determinants of broad‐scale flight strategies of migratory birds.