Consistent avoidance of human disturbance over large geographical distances by a migratory bird

Recent work on animal personalities has demonstrated that individuals may show consistent behaviour across situations and contexts. These studies were often carried out in one location and/or during short time intervals. Many animals, however, migrate and spend their life in several geographically distinct locations, and they may either adopt behaviours specific to the local environment or keep consistent behaviours over ecologically distinct locations. Long-distance migratory species offer excellent opportunities to test whether the animals maintain their personalities over large geographical scale, although the practical difficulties associated with these studies have hampered such tests. Here, we demonstrate for the first time consistency in disturbance tolerance behaviour in a long-distance migratory bird, using the common crane Grus grus as an ecological model species. Cranes that hatched in undisturbed habitats in Finland choose undisturbed migratory stop-over sites in Hungary, 1300-2000 km away from their breeding ground. This is remarkable, because these sites are not only separated by large distances, they also differ ecologically: the breeding sites are wooded bogs and subarctic tundra, whereas the migratory stop-over sites are temperate zone alkaline grasslands. The significance of our study goes beyond evolutionary biology and behavioural ecology: local effects on behaviour may carry over large distances, and this hitherto hidden implication of habitat selection needs to be incorporated into conservation planning.     

PKR-dependent autophagic degradation of herpes simplex virus type 1

The lysosomal pathway of autophagy is the major catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Recent studies have also shown that autophagy (xenophagy) may be used to degrade bacterial pathogens that invade intracellularly. However, it is not yet known whether xenophagy is a mechanism for degrading viruses. Previously, we showed that autophagy induction requires the antiviral eIF2alpha kinase signaling pathway (including PKR and eIF2alpha) and that this function of eIF2alpha kinase signaling is antagonized by the herpes simplex virus (HSV-1) neurovirulence gene product, ICP34.5. Here, we show quantitative morphologic evidence of PKR-dependent xenophagic degradation of herpes simplex virions and biochemical evidence of PKR and eIF2alpha-dependent degradation of HSV-1 proteins, both of which are blocked by ICP34.5. Together, these findings indicate that xenophagy degrades HSV-1 and that this cellular function is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. Thus, autophagy-related pathways are involved in degrading not only cellular constituents and intracellular bacteria, but also viruses.     

Constitutive endocytic cycle of the CB1 cannabinoid receptor

The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion ( approximately 85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.     

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.     

Lateral entorhinal cortex lesions rearrange afferents, glutamate receptors, increase seizure latency and suppress seizure-induced c-fos expression in the hippocampus of adult rat

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurones in chronic epilepsy. Here we analysed the effects of one-sided lateral EC (LEC) and temporoammonic (alvear) path lesion on the development and properties of 4-aminopyridine-induced seizures. Electroencephalography (EEG) analysis of freely moving rats identified that the lesion increased the latency of the hippocampal seizure significantly and decreased the number of brief convulsions. Seizure-induced neuronal c-fos expression was reduced in every hippocampal area following LEC lesion. Immunocytochemical analysis 40 days after the ablation of the LEC identified sprouting of cholinergic and calretinin-containing axons into the dentate molecular layer. Region and subunit specific changes in the expression of ionotropic glutamate receptors (iGluRs) were identified. Although the total amount of AMPA receptor subunits remained unchanged, GluR1(flop) displayed a significant decrease in the CA1 region. An increase in NR1 and NR2B N-methyl-d-aspartate (NMDA) receptor subunits and KA-2 kainate receptor subunit was identified in the deafferented layers of the hippocampus. These results further emphasize the importance of the lateral entorhinal area in the spread and regulation of hippocampal seizures and highlight the potential role of the rewiring of afferents and rearrangement of iGluRs in the dentate gyrus in hippocampal convulsive activity.     

In vitro binding of leukotriene B4 (LTB4) to human serum albumin: evidence from spectroscopic, molecular modeling, and competitive displacement studies

Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B4 (LTB4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB4 was enhanced fivefold in the presence of HSA. The red-shifted, hypochromic, and reduced vibrational fine structure of the ligand/protein UV absorption spectrum indicated complexation of the two molecules in solution. Results obtained from CD titration experiments were subjected to non-linear regression analysis to estimate the binding parameters (Ka = 6.7 x 10(4) M(-1), n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB4/HSA complex, suggesting the role of a high-affinity fatty acid HSA binding site in the leukotriene complexation. Molecular modeling calculations based on the crystal structure of HSA predicted that the long-chain fatty acid site that overlaps with drug binding site II in subdomain IIIA was the most likely binding location for LTB4. Using the drug site II-specific marker ligand rac-ibuprofen, this prediction was confirmed with induced-CD displacement measurements. To the authors' knowledge, the current study represents the first demonstration of binding of LTB4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.     

Synthesis of selective SRPK-1 inhibitors: novel tricyclic quinoxaline derivatives

SR protein-specific kinase-1 (SRPK-1) has been identified as a validated target for hepatitis B virus (HBV). A series of novel tricyclic quinoxaline derivatives was designed and synthesised as potential kinase inhibitory antiviral agents and was found to be active and selective for SRPK-1 kinase. Most of these novel compounds have drug-like properties according to experimentally determined LogP and LogS values.     

Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling

Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r=0.927 and r=0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. Chi2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.