Ligand-protein interactions in lysozyme investigated through a dual-resolution model.

A fully atomistic (AT) modeling of biological macromolecules at relevant length- and time-scales is often cumbersome or not even desirable, both in terms of computational effort required and a posteriori analysis. This difficulty can be overcome with the use of multiresolution models, in which different regions of the same system are concurrently described at different levels of detail. In enzymes, computationally expensive AT detail is crucial in the modeling of the active site in order to capture, for example, the chemically subtle process of ligand binding. In contrast, important yet more collective properties of the remainder of the protein can be reproduced with a coarser description. In the present work, we demonstrate the effectiveness of this approach through the calculation of the binding free energy of hen egg white lysozyme with the inhibitor di-N-acetylchitotriose. Particular attention is payed to the impact of the mapping, that is, the selection of AT and coarse-grained residues, on the binding free energy. It is shown that, in spite of small variations of the binding free energy with respect to the active site resolution, the separate contributions coming from different energetic terms (such as electrostatic and van der Waals interactions) manifest a stronger dependence on the mapping, thus pointing to the existence of an optimal level of intermediate resolution.

Molecular basis of tactile specialization in the duck bill.

Tactile-foraging ducks are specialist birds known for their touch-dependent feeding behavior. They use dabbling, straining, and filtering to find edible matter in murky water, relying on the sense of touch in their bill. Here, we present the molecular characterization of embryonic duck bill, which we show contains a high density of mechanosensory corpuscles innervated by functional rapidly adapting trigeminal afferents. In contrast to chicken, a visually foraging bird, the majority of duck trigeminal neurons are mechanoreceptors that express the Piezo2 ion channel and produce slowly inactivating mechano-current before hatching. Furthermore, duck neurons have a significantly reduced mechano-activation threshold and elevated mechano-current amplitude. Cloning and electrophysiological characterization of duck Piezo2 in a heterologous expression system shows that duck Piezo2 is functionally similar to the mouse ortholog but with prolonged inactivation kinetics, particularly at positive potentials. Knockdown of Piezo2 in duck trigeminal neurons attenuates mechano current with intermediate and slow inactivation kinetics. This suggests that Piezo2 is capable of contributing to a larger range of mechano-activated currents in duck trigeminal ganglia than in mouse trigeminal ganglia. Our results provide insights into the molecular basis of mechanotransduction in a tactile-specialist vertebrate.

Interaction of neuropeptide Y receptors (NPY1, NPY2 and NPY5) with somatostatin on somatostatin-induced feeding behaviour in neonatal chicken.

1. The present study was conducted to investigate whether brain somatostatin increases feed intake in neonatal chickens. The mediating role of neuropeptide Y receptors on feed intake induced by somatostatin was investigated. 2. In this study, seven experiments were designed, each with four treatment groups (n = 44 in each experiment). In Experiment 1, chicks received control solution and 0.5, 1 and 2 nmol of somatostatin through intracerebroventricular (ICV) injection. In experiments 2, 3 and 4, chickens were ICV injected with control solution and 1.25, 2.5 and 5 µg of B5063 (NPY1 receptor antagonist), SF22 (NPY2 receptor antagonist) and SML0891 (NPY5 receptor antagonist), respectively. In experiment 5, 6 and 7 chickens received ICV injection of B5063, SF22, SML0891, with a co-injection of + somatostatin, control solution and somatostatin. The cumulative feed intake was measured until 120 min post injection. 3. Somatostatin significantly increased feed intake in FD3 chicks. Both B5063 and SML0891 dose-dependently decreased feed intake compared with the control group, while SF22 led to a dose-dependent increase in feed intake. In addition, the hyperphagic effect of somatostatin significantly decreased with co-injection of B560 plus somatostatin (p < 0.05), but SF22 and SML0891 had no effect on feed intake induced by somatostatin in chicks (p > 0.05). 4. Based on the results of this study, it is likely that somatostatin increased feed intake and NPY1 receptor acts as a mediator in hyperphagic effect of somatostatin in neonatal chicks.

Down-regulation of cellular protein heme oxygenase-1 inhibits proliferation of avian influenza virus H9N2 in chicken oviduct epithelial cells.

The pathogenesis of H9N2 subtype avian influenza virus (AIV) infection in hens is often related to oviduct tissue damage. Our previous study suggested that H9N2 AIV induces cellular apoptosis by activating reactive oxygen species (ROS) accumulation and mitochondria-mediated apoptotic signalling in chicken oviduct epithelial cells (COECs). Heme oxygenase-1 (HO-1) is an inducible enzyme that exerts protective effects against oxidative stress and activated HO-1 was recently shown to have antiviral activity. To study the potential involvement of HO-1 in H9N2 AIV proliferation, the role of its expression in H9N2-infected COECs was further investigated. Our results revealed that H9N2 AIV infection significantly up-regulated the expression of HO-1 and that HO-1 down-regulation by ZnPP, a classical inhibitor of HO-1, could inhibit H9N2 AIV replication in COECs. Similarly, the small interfering RNA (siRNA)-mediated knockdown of HO-1 also markedly decreased the virus production in H9N2-infected COECs. In contrast, adenoviral-mediated over-expression of HO-1 concomitantly promoted H9N2 AIV replication. Taken together, our study demonstrated the involvement of HO-1 in AIV H9N2 proliferation, and these findings suggested that HO-1 is a potential target for inhibition of AIV H9N2 replication.

MiR-19b-3p Regulates MAPK1 Expression in Embryonic Fibroblasts from the Great Tit (Parus Major) Under Hypoxic Conditions.

BACKGROUND/AIMS: Genomic adaptations to high altitudes have been well studied in the last several years; however, the roles of microRNAs (miRNAs), which are essential modulators of a variety of genes and key cellular processes, have rarely been explored. Here, we explored the interactions between miRNAs and their target genes as an adaptation to high altitude in an avian species, the great tit (Parus major), which is widely distributed across the Eurasian continent at altitudes between 4500 m and sea level. Because the MAPK signaling pathway plays a crucial role in the hypoxia response in the great tit, we chose MAPK1 as a target candidate gene. METHODS: We established a great tit embryonic fibroblast line and subsequently studied the relationship between miRNA-19b-3p and MAPK1 in normoxia and hypoxia groups. Meanwhile, the great tit embryonic fibroblasts (GEFs) were treated or transfected with miR-19b-3p mimics, inhibitors, or si-MAPK1, and their proliferation was subsequently assessed using the MTT assay. The expression of the miRNAs and MAPK1 was measured by real-time PCR and Western blotting. RESULTS: We identified 14 miRNAs in the cardiac tissues of great tits that are related to hypoxia adaptation. MAPK1 binds only to miR-19b-3p of the 14 miRNAs predicted by both TargetScan and miRanda software. Specifically, we validated the computational prediction of miR-19b-3p binding to the 3'UTR of MAPK1 using a luciferase reporter assay. Our results show that miR-19b-3p promotes GEFs proliferation and up-regulates MAPK1 expression. Moreover, miR-19b-3p mimics and MAPK1 knockdown induce GEFs apoptosis and regulate the cell cycle under hypoxic conditions. CONCLUSIONS: Our study is the first to describe an important miRNA-mediated regulatory mechanism of high altitude adaptation in a non-model wild songbird and highlights the importance of studies on miRNA-mediated mechanisms of hypoxic adaptations in other animals.

Endocannabinoid hydrolases in avian HD11 macrophages identified by chemoproteomics: inactivation by small-molecule inhibitors and pathogen-induced downregulation of their activity.

The endocannabinoids (eCBs) are endogenous arachidonoyl-containing lipid mediators with important roles in host defense. Macrophages are first-line defenders of the innate immune system and biosynthesize large amounts of eCBs when activated. The cellular levels of eCBs are controlled by the activities of their biosynthetic enzymes and catabolic enzymes, which include members of the serine hydrolase (SH) superfamily. The physiologic activity of SHs can be assessed in a class-specific way using chemoproteomic activity-based protein profiling (ABPP) methods. Here, we have examined avian (chicken) HD11 macrophages, a widely used cell line in host-pathogen research, using gel-based ABPP and ABPP-multidimensional protein identification technology (MudPIT) to profile the changes in SH activities under baseline, chemical-inhibitor-treated, and pathogen-challenged conditions. We identified α/ß-hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH) as the principal SHs responsible for 2-arachidonoylglycerol (2AG) hydrolysis, thereby regulating the concentration of this lipid in HD11 cells. We further discovered that infection of HD11 macrophages by Salmonella Typhimurium caused the activities of these 2AG hydrolases to be downregulated in the host cells. ABHD6 and FAAH were potently inhibited by a variety of small-molecule inhibitors in intact live cells, and thus these compounds might be useful host-directed adjuvants to combat antimicrobial resistance in agriculture. 2AG was further shown to augment the phagocytic function of HD11 macrophages, which suggests that pathogen-induced downregulation of enzymes controlling 2AG hydrolytic activity might be a physiological mechanism to increase 2AG levels, thus enhancing phagocytosis. Together these results define ABHD6 and FAAH as 2AG hydrolases in avian macrophages that can be inactivated pharmacologically and decreased in activity during Salmonella Typhimurium infection.

Purification of glutathione S-transferase enzyme from quail liver tissue and inhibition effects of (3aR,4S,7R,7aS)-2-(4-((E)-3-(aryl)acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione derivatives on the enzyme activity.

The use of quail meat and eggs has made this animal important in recent years, with its low cost and high yields. Glutathione S-transferases (GST, E.C.2.5.1.18) are an important enzyme family, which play a critical role in detoxification system. In our study, GST was purified from quail liver tissue with 47.88-fold purification and 12.33% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by SDS-PAGE method and showed a single band. In addition, inhibition effects of (3aR,4S,7R,7aS)-2-(4-((E)-3-(aryl)acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7methanoisoindole-1,3(2H)-dion derivatives (1a-g) were investigated on the enzyme activity. The inhibition parameters (IC50 and Ki values) were calculated for these compounds. IC50 values of these derivatives (1a-e) were found as 23.00, 15.75, 115.50, 10.00, and 28.75 µM, respectively. Ki values of these derivatives (1a-e) were calculated in the range of 3.04 ± 0.50 to 131.50 ± 32.50 µM. However, for f and g compounds, the inhibition effects on the enzyme were not found.

Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2.

Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 µM for SmM, 3.58 µM for NM2a, 2.30 µM for NM2b, and 1.57 µM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.

Notch and Hedgehog in the thymus/parathyroid common primordium: Crosstalk in organ formation.

The avian thymus and parathyroids (T/PT) common primordium derives from the endoderm of the third and fourth pharyngeal pouches (3/4PP). The molecular mechanisms that govern T/PT development are not fully understood. Here we study the effects of Notch and Hedgehog (Hh) signalling modulation during common primordium development using in vitro, in vivo and in ovo approaches. The impairment of Notch activity reduced Foxn1/thymus-fated and Gcm2/Pth/parathyroid-fated domains in the 3/4PP and further compromised the development of the parathyroid glands. When Hh signalling was abolished, we observed a reduction in the Gata3/Gcm2- and Lfng-expression domains at the median/anterior and median/posterior territories of the pouches, respectively. In contrast, the Foxn1 expression-domain at the dorsal tip of the pouches expanded ventrally into the Lfng-expression domain. This study offers novel evidence on the role of Notch signalling in T/PT common primordium development, in an Hh-dependent manner.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

Semaphorin 5B is a repellent cue for sensory afferents projecting into the developing spinal cord.

During vertebrate development, centrally projecting sensory axons of the dorsal root ganglia neurons first reach the embryonic spinal cord at the dorsolateral margin. Instead of immediately projecting into the grey matter, they bifurcate and extend rostrally and caudally to establish the longitudinal dorsal funiculus during a stereotyped waiting period of approximately 48 h. Collateral fibres then extend concurrently across multiple spinal segments and project to their appropriate targets within the grey matter. This rostrocaudal extension of sensory afferents is crucial for the intersegmental processing of information throughout the spinal cord. However, the precise cues that prevent premature entry during the waiting period remain to be identified. Here, we show that semaphorin 5B (Sema5B), a member of the semaphorin family of guidance molecules, is expressed in the chick spinal cord during this waiting period and dorsal funiculus formation. Sema5B expression is dynamic, with a reduction of expression apparent in the spinal cord concomitant with collateral extension. We show that Sema5B inhibits the growth of NGF-dependent sensory axons and that this effect is mediated in part through the cell adhesion molecule TAG-1. Knockdown of Sema5B in the spinal cord using RNA interference leads to the premature extension of cutaneous nociceptive axons into the dorsal horn grey matter. These premature projections predominantly occur at the site of dorsal root entry. Our results suggest that Sema5B contributes to a repulsive barrier for centrally projecting primary sensory axons, forcing them to turn and establish the dorsal funiculus.

Design of peptidase-resistant peptide inhibitors of myosin light chain kinase.

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell-permeant peptide Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys (PIK, Peptide Inhibitor of Kinase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L-PIK in a biological milieu prompts for development of more stable L-PIK analogues for use as experimental tools in basic and drug-oriented biomedical research. Previously, we designed PIK1, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 , that was 2.5-fold more resistant to peptidases in human plasma in vitro than L-PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site-protected peptides based on L-PIK and PIK1 degradation patterns in human plasma as revealed by 1 H-NMR analysis. Implemented modifications increased half-live of the PIK-related peptides in plasma about 10-fold, and these compounds retained 25-100% of L-PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH2 , was identified as the most stable and effective L-PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin-induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell-permeant inhibitors of MLCK in cell culture-based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Ligands and Regulatory Modes of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) in Avians.

Nutrient and gene interaction is an important aspect of poultry metabolism that determines performance capacity. New technological tools in biochemistry and biotechnology make it possible to explore the molecular base of phenotypic characteristics of poultry production. Fats act as energy deposits in the poultry body and are an essential constituent of animal cell membranes. From a functional standpoint, it has been suggested that ingested lipids change liver fatty acid synthesis and other lipogenic enzymes by regulating mRNA synthesis. Nuclear hormone receptors are ligand-activated transcription factors that control several genes involved in lipid metabolism. The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of transcription factors. Three separate PPAR genes have been identified; they are known as α, δ, and γ. The most important metabolic effect of PPARγ in chicken is its task in adipogenesis. Reviewing the ligands of chicken PPARγ gene can be useful to a better understanding of PPARγ regulatory functions.

Cadherin-6B is required for the generation of Islet-1-expressing dorsal interneurons.

Cadherin-6B induces bone morphogenetic protein (BMP) signaling to promote the epithelial mesenchymal transition (EMT) in the neural crest. We have previously found that knockdown of Cadherin-6B inhibits both BMP signaling and the emigration of the early pre-migratory neural crest cells from the dorsal neural tube. In this study, we found that inhibition of BMP signaling in the neural tube, mediated by the ectopic expression of Smad-6 or Noggin, decreased the size of the Islet-1-positive dorsal cell population. Knockdown or loss of function of Cadherin-6B suppressed the generation of Islet-1-expressing cells in the dorsal neural tube, but not the Lim-1/2 positive dorsal cell population. Our results thus indicate that Cadherin-6B is necessary for the generation of Islet-1-positive dorsal interneurons, as well as the initiation of pre-migratory neural crest cell emigration.

Alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccDNA minichromosomes.

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV) replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg) RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9) and 27 (H3K27) in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.

The effects of actomyosin disruptors on the mechanical integrity of the avian crystalline lens.

PURPOSE: Actin and myosin within the crystalline lens maintain the structural integrity of lens fiber cells and form a hexagonal lattice cradling the posterior surface of the lens. The actomyosin network was pharmacologically disrupted to examine the effects on lenticular biomechanics and optical quality. METHODS: One lens of 7-day-old White Leghorn chickens was treated with 10 µM of a disruptor and the other with 0.01% dimethyl sulfoxide (vehicle). Actin, myosin, and myosin light chain kinase (MLCK) disruptors were used. The stiffness and the optical quality of the control and treated lenses were measured. Western blotting and confocal imaging were used to confirm that treatment led to a disruption of the actomyosin network. The times for the lenses to recover stiffness to match the control values were also measured. RESULTS: Disruptor-treated lenses were significantly less stiff than their controls (p≤0.0274 for all disruptors). The disruptors led to changes in the relative protein amounts as well as the distributions of proteins within the lattice. However, the disruptors did not affect the clarity of the lenses (p≥0.4696 for all disruptors), nor did they affect spherical aberration (p = 0.02245). The effects of all three disruptors were reversible, with lenses recovering from treatment with actin, myosin, and MLCK disruptors after 4 h, 1 h, and 8 min, respectively. CONCLUSIONS: Cytoskeletal protein disruptors led to a decreased stiffness of the lens, and the effects were reversible. Optical quality was mostly unaffected, but the long-term consequences remain unclear. Our results raise the possibility that the mechanical properties of the avian lens may be actively regulated in vivo via adjustments to the actomyosin lattice.

25-Hydroxycholecalciferol Enhances Male Broiler Breast Meat Yield through the mTOR Pathway.

BACKGROUND: In recent years, there has been a growing body of evidence indicating that replacing cholecalciferol (vitamin D3) with 25-hydroxycholecalciferol [25(OH)D3] through dietary supplementation enhances breast meat yield in broiler chickens. However, the underlying molecular mechanisms are still unknown. OBJECTIVE: We investigated the effect of 25(OH)D3 on male broiler growth performance (body weight, feed intake, feed conversion ratio, and breast meat yield), muscle protein synthesis, and the potential underlying molecular mechanisms. METHODS: Male Cobb 500 broiler chickens were divided into 4 body weight-matched groups and received a control diet with normal cholecalciferol (2760 IU/kg feed) for 42 d, a diet with high concentrations of cholecalciferol (5520 IU/kg feed) for 42 d, or a diet with 25(OH)D3 (5520 IU/kg feed) for 42 d (HyD-42). A fourth group consumed the HyD-42 for 21 d and then control feed for 21 d (HyD-21) (n = 360 birds, 12 replicates/treatment). Food and clean water were available for ad libitum consumption. At the end of the 42-d experiment, protein turnover was measured by phenylalanine flooding dose. Breast muscle tissues were collected and protein synthesis-related gene and protein expression were measured by real time polymerase chain reaction and Western blot, respectively. Functional studies were performed in vitro with the use of a quail myoblast (QM7) cell line. QM7 cells were treated with 2 doses (1 nM and 10 nM) of cholecalciferol or 25(OH)D3 alone or in combination with 100 nM rapamycin, and cell proliferation was determined by cell proliferation assay. Protein synthesis-related gene and protein expression were also determined. RESULTS: The HyD-42 increased 25(OH)D3 circulating concentrations by 126% (P < 0.05), enhanced breast meat yield (P < 0.05), and increased the fractional rate of protein synthesis by 3-fold (P < 0.05) compared with the control diet. Molecular analyses revealed that breast muscle from chickens consuming the HyD-42 expressed significantly higher concentrations of vitamin D receptor (VDR), phospho mechanistic target of rapamycin(Ser2481), phospho ribosomal P70 S6 kinase (RPS6K)(Thr421/Ser424), and antigen Ki-67 (Ki67) compared with the other groups. In line with the in vivo data, in vitro functional studies showed that cells treated with 25(OH)D3 for 24 h had increased VDR expression, and activated the mechanistic target of rapamycin (mTOR)/S6 kinase (S6K) pathway, enhanced Ki67 protein concentrations, and induced QM7 cell proliferation compared with untreated or cholecalciferol-treated cells. Blocking the mTOR pathway with rapamycin reversed these effects. CONCLUSION: Taken together, our findings provide evidence that the effects of 25(OH)D3 on male broiler breast muscle are likely mediated through the mTOR-S6K pathway.

Activation of muscarinic acetylcholine receptors elevates intracellular Ca(2+) concentrations in accessory lobe neurons of the chick.

Accessory lobes are protrusions located at the lateral sides of the spinal cord of chicks and it has been proposed that they play a role as a sensory organ for equilibrium during walking. We have reported that functional neurons exist in the accessory lobe. As there is histological evidence that synaptic terminals of cholinergic nerves exist near the somata of accessory lobe neurons, we examined the effects of acetylcholine on changes in intracellular Ca2+ concentrations ([Ca2+]i), as an index of cellular activities. Acetylcholine (0.1-100 µM) caused a transient rise in the [Ca2+]i. Acetylcholine-evoked [Ca2+]i rises were observed in the absence of extracellular Ca2+, and they were abolished in the presence of cyclopiazonic acid, an inhibitor of Ca2+-ATPase of intracellular Ca2+ stores or atropine, a muscarinic receptor antagonist. mRNAs coding M3 and M5 isoforms of the muscarinic receptors were detected in accessory lobes by the RT-PCR. These results indicate that chick accessory lobe neurons express functional muscarinic acetylcholine receptors, and that acetylcholine stimulates Ca2+ mobilization from intracellular Ca2+ stores, which elevates the [Ca2+]i in the somata of accessory lobe neurons, through activation of these receptors. Cholinergic synaptic transmission to the accessory lobe neurons may regulate some cellular functions through muscarinic receptors.

FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools.

The size, composition and functioning of the spinal cord is likely to depend on appropriate numbers of progenitor and differentiated cells of a particular class, but little is known about how cell numbers are controlled in specific cell cohorts along the dorsoventral axis of the neural tube. Here, we show that FatJ cadherin, identified in a large-scale RNA interference (RNAi) screen of cadherin genes expressed in the neural tube, is localised to progenitors in intermediate regions of the neural tube. Loss of function of FatJ promotes an increase in dp4-vp1 progenitors and a concomitant increase in differentiated Lim1(+)/Lim2(+) neurons. Our studies reveal that FatJ mediates its action via the Hippo pathway mediator Yap1: loss of downstream Hippo components can rescue the defect caused by loss of FatJ. Together, our data demonstrate that RNAi screens are feasible in the chick embryonic neural tube, and show that FatJ acts through the Hippo pathway to regulate cell numbers in specific subsets of neural progenitor pools and their differentiated progeny.

TCDD­induced chick cardiotoxicity is abolished by a selective cyclooxygenase­2 (COX­2) inhibitor NS398.

Halogenated aromatic hydrocarbons, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are known to cause severe heart defects in avian species. However, the mechanism of TCDD-induced chick cardiovascular toxicity is unclear. In this study, we investigated cyclooxygenase-2 (COX-2) as a possible mechanism of TCDD-induced cardiotoxicity. Fertile chicken eggs were injected with TCDD and a COX-2 selective inhibitor, NS398, and we investigated chick heart failure on day 10. We found that the chick heart to body weight ratio and atrial natriuretic factor mRNA expression were increased, but this increase was abolished with treatment of NS398. In addition, the morphological abnormality of an enlarged ventricle resulting from TCDD exposure was also abolished with co-treatment of TCDD and NS398. Our results suggested that TCDD-induced chick heart defects are mediated via the nongenomic pathway and that they do not require the genomic pathway.