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.

Targeted disruption of TORC1 retards young squab growth by inhibiting the synthesis of crop milk protein in breeding pigeon (Columba livia).

This study was conducted to explore the regulatory role of the target of rapamycin complex 1 (TORC1) signaling pathway in crop milk synthesis in breeding pigeons (Columba livia). Three groups of breeding pigeons in the lactation period (n = 30 pairs/group) were respectively injected with rapamycin (RAPA, a specific inhibitor of the target of rapamycin complex) at doses of 0 (vehicle, control), 0.6, or 1.2 mg/kg body weight (BW)/day via the wing vein for 7 days. The average daily feed intake (ADFI) and BW of the breeding pigeons and the BW of young squabs were respectively recorded throughout the experimental period. The breeding pigeons were sacrificed to collect their crop tissues, crop milk, and serum on the eighth day of the experiment. The results showed that neither 0.6 nor 1.2 mg/kg BW RAPA injection affected BW loss or ADFI in breeding pigeons (P > 0.05), while crop thickness and crop relative weight were significantly decreased (P < 0.05) in the 1.2 mg/kg BW rapamycin-injected group. Simultaneously, RAPA (especially at 1.2 mg/kg BW) decreased the crude protein, αs1-casein, αs2-casein, ß-casein, and amino acid contents (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Lys, His, Arg, and Pro) of crop milk (P < 0.05) and the concentrations of albumin, total protein, and uric acid in the serum of breeding pigeons (P < 0.05). Additionally, the expression of TORC1 pathway-related proteins (TORC1, S6K1, S6, 4EBP1, and eIF4E) was downregulated in the crop tissues of breeding pigeons by 0.6 or 1.2 mg/kg BW/day RAPA injection (P < 0.05). Accordingly, the average daily gain (ADG) of young squabs declined, and the mortality rate increased significantly (P < 0.05). Together, the results showed that RAPA reduced protein and amino acid levels in the crop milk of breeding pigeons and retarded young squab growth, suggesting a crucial role of TORC1 in crop milk synthesis in breeding pigeons.

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.

Postmortem role of calpain-11 in ostrich skeletal muscle.

The postmortem calpain-11 role in ostrich muscle was investigated. Pairs of ostrich muscle (Iliotibialis cranialis) were excised from 32 ostrich carcasses in 3-h postmortem and randomly assigned into four treatments. The muscle was cut into 2.5-cm thick meat cores. The cores were incubated in 30 mM CaCl2, 30 mM EDTA, 90 mM NaCl, or control. The cores from the left-side carcasses were sampled after 0, 1, 2, and 3 days of incubation at 5 °C, while the right-side meat cores were taken at 1-day and 3-day incubation for shear force measurements. The results showed that the decrease in unautolyzed and total activities of calpain-11, desmin content and shear force was more rapid in CaCl2-incubated samples than in control, NaCl- and EDTA-incubated samples. Thus, present results suggest that in the absence of calpain-1, calpain-11 with an extensive activation by adding exogenous Ca2+ could enhance the postmortem proteolysis and tenderization of ostrich muscle.

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.

Localized delivery of miRNAs targets cyclooxygenases and reduces flexor tendon adhesions.

The formation of adhesions during healing of an injured tendon remains a difficult problem in clinical practice. Local anti-inflammation gene delivery provides high local gene concentration, reduces the inflammatory response of the injured tendon microenvironment, and decreases systemic side effects to enhance in vivo efficacy. In this study, we designed a novel local sustained gene delivery system by using cyclooxygenase (COX-1 and COX-2)-engineered miRNA plasmid/nanoparticles embedded in hyaluronic acid (HA) hydrogel to reduce flexor tendon adhesions. The local sustained gene delivery system significantly downregulates COX-1 and COX-2 expression in the tendon tissue and the surrounding subcutaneous tissue. More importantly, this plasmid/nanoparticle hydrogel system significantly reduced tissue adhesion formation. This approach offers an effective therapeutic strategy to reduce tendon adhesions by directly targeting the down-regulation of COX-1 and COX-2 expression within the microenvironment of the injured tendon. STATEMENT OF SIGNIFICANCE: A local sustained gene delivery system was developed to regulate the expression of targeted genes in the specific time and location for tendon adhesion treatment. The engineered miRNA plasmid/nanoparticles embedded in hyaluronic acid hydrogel were synthesized to downregulate the expression of cyclooxygenases in the tendon tissue during the early stage of tendon healing with inflammatory response. This plasmid/nanoparticle hydrogel system offers an effective therapeutic strategy to attenuate the formation of tendon adhesion through direct downregulation of COX-1 and COX-2 expression within the microenvironment of the injured tendon.

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.

Erbin and ErbB2 play roles in the sexual differentiation of the song system nucleus HVC in bengalese finches (Lonchura Striata var. domestica).

Song control nuclei have distinct sexual differences in songbirds. However, the mechanism that underlies the sexual differentiation of song nuclei is still not well understood. Using a combination of anatomical, pharmacological, genetic, and behavioral approaches, the present study investigated the role of erbb2 (a homolog of the avian erythroblastic leukemia viral oncogene homolog 2) and the erbb2-interacting gene, erbin, in the sexual differentiation of the song nucleus HVC in the Bengalese finch. We first found that both erbin and erbb2 were expressed in the developing HVC at posthatch day (PHD) 15 in a male-biased fashion using qRT-PCR and in situ hybridization. Following the addition of a pharmaceutical inhibitor of the ErbB2 signaling pathway to the culture medium, cell proliferation in the cultured ventricle zone (VZ) that overlies the developing HVC decreased significantly. After the injection of erbin- or erbb2-interfering lentiviruses into the HVC and its overlying VZ at PHD 15, the cell proliferation in the VZ at PHD 24, the number of the differentiated neurons (Hu+ /BrdU+ or NeuN+ /BrdU+ ) in the HVC at PHD 31 or PHD 130, and the number of RA-projecting cells at PHD 130 all decreased significantly. Additionally, the adult songs displayed serious abnormalities. Finally, 173 male-biased genes were expressed in the developing HVC at PHD 15 using cDNA microarrays, of which 27.2% were Z-linked genes and approximately 20 genes were involved in the Erbin- or ErbB2-related signaling pathways. Our results provide some specific genetic factors that contribute to neurogenesis and sex differentiation in a song nucleus of songbirds. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 15-38, 2018.

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.

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.

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.

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.

Excess Manganese-Induced Apoptosis in Chicken Cerebrums and Embryonic Neurocytes.

There were many studies about the effect of excess manganese (Mn) on nervous system apoptosis; however, Mn-induced apoptosis in chicken cerebrums and embryonic neurocytes was unclear. The purpose of this study was to investigate the effect of excess Mn on chicken cerebrum and embryonic neurocyte apoptosis. Seven-day-old Hyline male chickens were fed either a commercial diet or three levels of manganese chloride (MnCl2)-added commercial diets containing 600-, 900-, and 1800-mg/kg-Mn diet, respectively. On the 30th, 60th, and 90th days, cerebrums were collected. Fertilized Hyline chicken eggs were hatched for 6-8 days and were selected. Embryonic neurocytes with 0, 0.5, 1, 1.5, 2, 2.5, and 3 mM Mn were collected and were cultured for 12, 24, 36, and 48 h, respectively. The following research contents were performed: superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities; tumor protein p53 (p53), B cell lymphoma-2 (Bcl-2), B cell lymphoma extra large (Bcl-x), Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), fas, and caspase-3 messenger RNA (mRNA) expression; and morphologic observation. The results indicated that excess Mn inhibited SOD and T-AOC activities; induced p53, Bax, Bak, fas, and caspase-3 mRNA expression; and inhibited Bcl-2 and Bcl-x mRNA expression in chicken cerebrums and embryonic neurocytes. There were dose-dependent manners on all the above factors at all the time points and time-dependent manners on SOD activity of 1800-mg/kg-Mn group, T-AOC activity, and apoptosis-related gene mRNA expression in all the treatment groups in chicken cerebrums. Excess Mn induced chicken cerebrum and embryonic neurocyte apoptosis.

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.

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.

Dopamine D3 receptors modulate the rate of neuronal recovery, cell recruitment in Area X, and song tempo after neurotoxic damage in songbirds.

Songbirds, like humans, learn vocalizations and their striatum recruits new neurons in adulthood. Injury in striatal vocal nucleus Area X, involved in song learning and production in songbirds, is followed by massive regeneration. The newborn neurons arise from the subventricular zone (SVZ) rich in dopamine D3 receptors (D3Rs). The aim of this study was to investigate whether the D3Rs affect the rate of neuronal recovery in Area X. Male zebra finches (Taeniopygia guttata) received bilateral neurotoxic lesion of Area X and were implanted with osmotic minipumps containing D3R agonist 7-OH-DPAT, antagonist U99194, or saline. Treatment with 7-OH-DPAT but not U99194 led to significant reduction of lesion size and increased numbers of migrating neuroblasts and newborn cells in the Area X. These cells were detected in the lesion border as well as the lesion center. Lesion also led to increased mRNA expression of the D3Rs in the neurogenic SVZ and in the nucleus robustus arcopallialis (RA) involved in song production. Moreover, lesion alone prolonged the song duration and this may be facilitated by D3Rs in RA. Parallel lesion and stimulation of D3Rs prolonged it even more, while blocking of D3Rs abolished the lesion-induced effect. These data suggest that D3R stimulation after striatal injury accelerates the striatal recovery and can cause behavioral alterations.

Diencephalic and septal structures containing the avian vasotocin receptor (V1aR) involved in the regulation of food intake in chickens, Gallus gallus.

Recently, it was found that the avian central vasotocin receptor (V1aR) is associated with the regulation of food intake. To identify V1aR-containing brain structures regulating food intake, a selective V1aR antagonist SR-49059 that induced food intake was administrated intracerebroventricularly in male chickens followed by detection of brain structures using FOS immunoreactivity. Particularly, the hypothalamic core region of the paraventricular nucleus, lateral hypothalamic area, dorsomedial hypothalamic nucleus, a subnucleus of the central extended amygdalar complex [dorsolateral bed nucleus of the stria terminalis], medial septal nucleus and caudal brainstem [nucleus of the solitary tract] showed significantly increased FOS-ir cells. On the other hand, the supraoptic nucleus of the preoptic area and the nucleus of the hippocampal commissure of the septum showed suppressed FOS immunoreactivity in the V1aR antagonist treatment group. Further investigation revealed that neuronal activity of arginine vasotocin (AVT-ir) magnocellular neurons in the supraoptic nucleus, preoptic periventricular nucleus, paraventricular nucleus and ventral periventricular hypothalamic nucleus and most likely corticotropin releasing hormone (CRH-ir) neurons in the nucleus of the hippocampal commissure were reduced following the antagonist treatment. Dual immunofluorescence labeling results showed that perikarya of AVT-ir magnocellular neurons in the preoptic area and hypothalamus were colabeled with V1aR. Within the nucleus of the hippocampal commissure, CRH-ir neurons were shown in close contact with V1aR-ir glial cells. Results of the present study suggest that the V1aR plays a role in the regulation of food intake by modulating neurons that synthesize and release anorectic neuropeptides in the avian brain.

Inhibition of TrkB limits development of the zebra finch song system.

Large sexual dimorphisms exist in the zebra finch song system. Masculinization may be mediated by both estradiol and expression of one or more Z-genes (males: ZZ; females: ZW). Roles of the Z-gene tyrosine kinase B (TrkB) in HVC in masculinizing both HVC and one of its targets the robust nucleus of the arcopallium (RA), were tested using siRNA administration in juvenile males at two ages (post-hatching days 15-17 or 25-27). Birds were euthanized 10 days later. Potential interactions or additive effects with estradiol were evaluated by treating males with the estrogen synthesis inhibitor fadrozole. Females treated with estradiol were also exposed to the siRNA at the later age. Local inhibition of TrkB in males of both ages reduced the volume of HVC, an effect due to a change in cell number and not cell size. In the older males, in which the treatment spanned the period when the projection from HVC to RA grows, TrkB inhibition reduced the volume of RA and the relative number of cells within it. TrkB siRNA in HVC decreased the volume of and soma size in the RA of females, and the projection from HVC to RA in both sexes. Estradiol in females masculinized various aspects of the song system, and its effect in masculinizing the volume of RA was decreased by TrkB inhibition. However, effects of fadrozole in males were limited. The data indicate that TrkB is involved in masculinizing the song system, but for most measures it probably does not work in concert with E2.

Knockdown of Lmo7 inhibits chick myogenesis.

The multifunctional protein Lmo7 has been implicated in some aspects of myogenesis in mammals. Here we studied the distribution and expression of Lmo7 and the effects of Lmo7 knockdown in primary cultures of chick skeletal muscle cells. Lmo7 was localized within the nuclei of myoblasts and at the perinuclear region of myotubes. Knockdown of Lmo7 using siRNA specific to chick reduces the number and width of myotubes and the number of MyoD positive-myoblasts. Both Wnt3a enriched medium and Bio, activators of the Wnt/beta-catenin pathway, could rescue the effects of the Lmo7 knockdown suggesting a crosstalk between the Wnt/beta-catenin and Lmo7-mediated signaling pathways. Our data shows a role of Lmo7 during the initial events of chick skeletal myogenesis, particularly in myoblast survival.