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The present study investigated the effect of intrathecal injection of (RS)-2-α-amino-3-(3-hydroxy-5-tbutylisoxazol-4-yl) propanoic acid (ATPA), a selective agonist to kainate receptor, on nociception in rats. Intrathecal administration of 1, 4 and 10 nmol of ATPA induced dose-dependent increases in the hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation in rats. Pretreatment with intrathecal injection of 300 μg of concanavalin A (ConA) to block the desensitization of kainate receptors enhanced and prolonged the anti-nociceptive effect induced by intrathecal injection of ATPA. The results suggest that the pre-synaptic kainate receptor in the primary afferent terminals is involved in the transmission of nociceptive information in dorsal horn of the spinal cord in rats. Furthermore, blocking the desensitization of kainate receptor enhanced and prolonged the ATPA-induced anti-nociceptive effects. [Copyright &y& Elsevier]

Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAV infection in different species. Recently, nanobodies, which show advantages of easy gene editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nano-bodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can crossreact with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen and dilutions of the fusion and testing sera were 100 ng/well, 1:4000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. In addition, we found that the cELISA and hemagglutination inhibition test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion-based cELISA is an ideal method for monitoring IAV infection in different species. [ABSTRACT FROM AUTHOR]

Abstract: Rationale: The long-term intermittent hypoxia (LTIH) that characterizes sleep-disordered breathing impairs spatial learning and increases oxidative stress in rodents. We hypothesized that LTIH activated brain NADPH oxidase, which served as a critical source of superoxide in the oxidation injury, and that apocynin might attenuate LTIH-induced spatial learning deficits by reducing LTIH-induced NADPH oxidase expression. Objective: To investigate the effects of apocynin on spatial learning and oxidative responses to LTIH in rats. Methods: Forty healthy male Sprague–Dawley (SD) rats were randomly divided into four groups of 10 each: a LTIH group, an apocynin-treated LTIH group, a sham LTIH group and an apocynin-treated sham group. Spatial learning in each group was assessed with the Morris water maze test. RT-PCR and Western blot were used to examine mRNA and protein expression of NADPH oxidase subunit p47phox and p22phox in the hippocampus region. The level of MDA and SOD were detected by colorimetric method. The terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) method was used to display the apoptotic cells of the hippocampus tissue. Results: Apocynin treatment prevented LTIH-induced decreases in spatial learning during the Morris water maze as well as LTIH-induced decrease in SOD levels. In untreated animals, LTIH exposure was related to increase of MDA levels in comparison to sham LTIH animals, and apocynin-treated animal exposure to LTIH showed reduction in MDA levels. Increases in hippocampus NADPH oxidase subunit p47phox mRNA and protein expression were observed in LTIH-exposed animals; there was no statistical difference of p47phox mRNA and protein expression between LTIH group and apocynin treatment group. Treatment with apocynin significantly ameliorated cell apoptosis in LTIH-exposed animals. Conclusion: These results indicate that apocynin attenuates LTIH-induced spatial learning deficits and mitigates LTIH-induced oxidative stress through multiple beneficial effects on oxidant pathways. NADPH oxidase up-expression is closely associated with oxidative processes in LTIH rats, and inhibition of NADPH oxidase activity may hopefully serve as a useful strategy for cognitive function impairment from chronic intermittent hypoxia. [Copyright &y& Elsevier]

Members of the bone morphogenetic protein (BMP) and T-box gene families play several critical roles in the early embryonic development and tissue homeostasis. Although BMP proteins are the upstream regulators of T-box genes, few studies have investigated the molecular mechanisms between these two protein families. Here, we report that Tbx6 interacts directly with Smad6, an inhibitory Smad that antagonizes the BMP signal. This interaction is mediated through the Mad homology 2 (MH2) domain of Smad6 and residues 90-180 of Tbx6. We demonstrate that Smad6 facilitates the degradation of Tbx6 protein through recruitment of Smurf1, a ubiquitin E3 ligase. Consequently, Smad6 reduces Tbx6-mediated Myf-5 gene activation. Furthermore, specific knockdown of endogenous Smad6 and Smurf1 by small interfering RNA increases the protein levels of Tbx6 and enhance the expression of Tbx6 target genes. Collectively, these findings reveal that Smad6 serves as a critical mediator of BMP signal via a functional interaction with Tbx6, thus regulating the activation of Tbx6 downstream genes during cell differentiation. [ABSTRACT FROM AUTHOR]

Members of the Swi2/Snf2 (switch/sucrose non-fermentable) family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein that mediates RNAP recycling during transcription. It is known that the ATPase activity of RapA is stimulated by its interaction with RNAP. It is not known, however, how the RapA-RNAP interaction activates the enzyme. Previously, we determined the crystal structure of RapA. The structure revealed the dynamic nature of its N-terminal domain (Ntd), which prompted us to elucidate the solution structure and activity of both the full-length protein and its Ntd-truncated mutant (RapAΔN). Here, we report the ATPase activity of RapA and RapAΔN in the absence or presence of RNAP and the solution structures of RapA and RapAΔN either ligand-free or in complex with RNAP. Determined by small-angle x-ray scattering, the solution structures reveal a new conformation of RapA, define the binding mode and binding site of RapA on RNAP, and show that the binding sites of RapA and σ70 on the surface of RNAP largely overlap. We conclude that the ATPase activity of RapA is inhibited by its Ntd but stimulated by RNAP in an allosteric fashion and that the conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling. These and previous findings outline the functional cycle of RapA, which increases our understanding of the mechanism and regulation of Swi2/Snf2 proteins in general and of RapA in particular. The new structural information also leads to a hypothetical model of RapA in complex with RNAP immobilized during transcription. [ABSTRACT FROM AUTHOR]

Elevated expression of family with sequence similarity 83 member D (Fam83D) has been found in various cancers; however, its role in pancreatic adenocarcinoma (PDAC) remains unclear. The current study was designed to elucidate the roles of Fam83D in pancreatic cancer. The level of Fam83D was detected in PDAC tissues and adjacent no-tumorous tissues. Effects of Fam83D on proliferation, glycolysis and gemcitabine (GEM) sensitivity of pancreatic cancer cells were examined. Fam83D was overexpressed in PDAC and associated with clinical stage, metastatic status and survival rates of PDAC patients. Function study showed that Fam83D knockdown (KD) caused inhibited proliferation, suppressed mitochondrial respiration capacity, reduced aerobic glycolysis, and down-regulation of nuclear β-catenin, proto-oncogene C-Myc, and lactate dehydrogenase A (LDHA). Fam83D KD enhanced the sensitivity of PDAC cells to GEM in vitro and in vivo. On the contrary, Fam83D overexpression displayed reverse effects on PDAC cells. Moreover, the Wnt/β-catenin inhibitor abolished the effects of Fam83D overexpression in PDAC cells. the current data suggest that enhanced Fam83D expression contributes to PDAC progression and the development of chemoresistance through the Wnt/β-catenin signaling. [ABSTRACT FROM AUTHOR]