Long‐term variation in the distribution of the White‐bellied Sea‐Eagle (Haliaeetus leucogaster) across Australia

Abstract The White‐bellied Sea‐Eagle Haliaeetus leucogaster (Accipitridae) is widespread within Australia. However, in a number of states it is thought to be declining locally in response to human induced disturbance. Here we analyse the Australian Bird Atlas data to identify the extent and pattern of change in range and density of the species between three Atlas Periods (1901–1976, 1977–1981 and 1998–2001) using a new standardized frequency measure, the Occupancy Index (OI) for 1° blocks (approx. 100 km²) across the continent. At the continental scale, there was no significant difference in the spatial extent of occupancy between Atlas Periods. However, there were considerable changes in frequency and range extent between defined regions, and there were distinct differences in the pattern of change in OI between coastal and inland blocks over time. Coastal blocks showed much more change than inland blocks, with a clear increase in the use of coastal blocks, accompanied by a decrease in inland blocks, during the 1977–1981 Atlas Period, relative to both other Atlas Periods. While there were slight (and not statistically significant) trends for OI to increase in areas containing dams, and to decrease in urbanized coastal areas, the over‐riding factor associated with distributional shifts and frequency changes was apparently climatic fluctuation (the 1977–1981 period showing the influence of El Niño associated drought). Within this study, the impression of abundance was strongly dependent on both the temporal and spatial scale of analysis. This highlights the importance of large‐scale analysis in interpreting change in distribution and abundance of widespread species.     

Collagen of fibrocartilage: a distinctive molecular phenotype in bovine meniscus

A fluorogenic substrate for plasmin, CBZ-Gly-Pro-Arg-AEC, has been synthesized and used to develop a new sensitive photometric and fluorometric assay of plasminogen activator activity. The fluorescence intensity of free AEC at 460 nm is about 3 orders of magnitude higher than that of acyl-AEC. The release of AEC from the peptidyl derivative was monitored fluorometrically after extraction of free AEC in ethylacetate. Under such conditions, the Km was 0.16 mM. This method was used to monitor the activity of plasminogen activator synthetized by fibroblastic cells (BHK 21 C 13) either released in the supernatants or cell-associated.     

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1^−/− mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1^+/+ and Npc1^−/− microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1^−/− microglia is ~ 60% lower than in Npc1^+/+ microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1^−/− and Npc1^+/+ neurons in microglia-neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1^−/− microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia-neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: selective stimulation of PKD Ser⁷⁴⁸ autophosphorylation by Gαq

Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4⁻). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser⁷⁴⁴, a prominent and rapid PKC transphosphorylation site, and Ser⁷⁴⁸, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4⁻ potently induced PKD activation and Ser⁷⁴⁴ and Ser⁷⁴⁸ phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser⁷⁴⁸ phosphorylation induced by AlF4⁻, and largely abolished Ser⁷⁴⁴ phosphorylation. In contrast, Ser⁷⁴⁸ phosphorylation was almost completely intact, and Ser⁷⁴⁴ phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser⁷⁴⁸ phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser⁷⁴⁸.     

Staurosporine-induced apoptosis presents with unexpected cholinergic effects in a differentiated neuroblastoma cell line

Apoptosis of cholinergic neurons is one of the core hallmarks of Alzheimer’s disease. SH-SY5Y neuroblastoma cells differentiated to the cholinergic phenotype were exposed to 100 nM staurosporine. Over a treatment period of 24 h, the pro- and anti-apoptotic factors, caspase-3 and Bcl-2, as well as LDH release as a measure of cell viability, were assessed in conjunction with the number of apoptotic cells by means of fluorescence-activated cell sorting. Caspase-3 activity and LDH release increased by 30% and 20% over controls, respectively, while Bcl-2 levels rose by 200% over controls. Furthermore, staurosporine treatment resulted in decreased acetylcholinesterase (AChE) enzymatic activity and decreased protein levels of the AChE splice variant tailed AChE (AChE-T). Only a slight increase in levels of readthrough AChE (AChE-R) was observed. Likewise, staurosporine reduced levels and activity of the cholinergic players choline acetyltransferase and high affinity choline uptake. The present study demonstrates that treatment with staurosporine leads to apoptotic events, which, however, are not reflected in the increased AChE activity and the alterations of AChE isoforms expression that are usually seen in apoptotic conditions. The effects of various additional phosphorylation inhibitors on AChE activity suggest that these unexpected cholinergic effects, firstly, are linked to the impact of staurosporine on phosphorylation and, secondly, reveal themselves in a first phase of cellular adaption that precedes neurotoxicity and subsequent cell death.     

The neural adhesion molecule L1CAM confers chemoresistance in human glioblastomas

Glioblastoma multiforme (GBM) represents the most common and malignant brain tumor. GBM tissues exhibit elevated expression of the transforming growth factor-beta1 (TGF-β1) and the adhesion molecule L1CAM. This study investigated the mechanism of L1CAM regulation in GBM cells and its role in the mediation of chemoresistance. L1CAM expression levels varied in GBM cells being highest in A172 cells and low in T98G cells. Inhibition of TGF-β1 signaling in A172 cells reduced L1CAM expression and vice versa stimulation with exogenous TGF-β1 led to upregulation of L1CAM in T98G cells. Additionally, TGF-β1 and L1CAM expression increased during differentiation of glioma stem-like cells. L1CAM expressing GBM cells and differentiated glioma stem-like cells showed a reduced apoptotic response after treatment with the chemotherapeutic drug temozolomide. Accordingly, siRNA-mediated knock-down of L1CAM in A172 cells and differentiated glioma stem-like cells increased chemosensitivity, whereas overexpression of L1CAM in T98G cells and glioma spheroids diminished the apoptotic response. Elevated L1CAM expression caused a diminished expression of caspase-8 in GBM and differentiated glioma stem-like cells. These data show that TGF-β1 dependent upregulation of L1CAM expression in GBM cells leads to the downregulation of caspase-8 and apoptosis resistance pointing to L1CAM as potential target for improved therapy of GBM patients.     

Limitations and mechanisms influencing the migratory performance of soaring birds

Migration is costly in terms of time, energy and safety. Optimal migration theory suggests that individual migratory birds will choose between these three costs depending on their motivation and available resources. To test hypotheses about use of migratory strategies by large soaring birds, we used GPS telemetry to track 18 adult, 13 sub‐adult and 15 juvenile Golden Eagles Aquila chrysaetos in eastern North America. Each age‐class had potentially different motivations during migration. During spring, the migratory performance (defined here as the directness of migratory flight) of adults was higher than that of any other age‐classes. Adults also departed earlier and spent less time migrating. Together, these patterns suggest that adults were primarily time‐limited and the other two age‐classes were energy‐limited. However, adults that migrated the longest distances during spring also appeared to take advantage of energy‐conservation strategies such as decreasing their compensation for wind drift. During autumn, birds of all age‐classes were primarily energy‐minimizers; they increased the length of stopovers, flew less direct routes and migrated at a slower pace than during spring. Nonetheless, birds that departed later in autumn flew more directly, indicating that time limitations may have affected their decision‐making. During both seasons, juveniles had the lowest performance, sub‐adults intermediate performance and adults the highest performance. Our results show age‐ and seasonal variation in time and energy‐minimization strategies that are not necessarily exclusive of one another. Beyond time and energy, a complex suite of factors, including weather, experience and navigation ability, influences migratory performance and decision‐making.