Past studies have proposed a role for the hippocampus in the rapid encoding of context memories. Despite this, there is little data regarding the molecular processes underlying the stable formation of a context representation that occurs in the time window established through such behavioral studies. One task that is useful for investigating the rapid encoding of context is contextual fear conditioning (CFC). Behavioral studies demonstrate that animals require approximately 30 s of exploration prior to a footshock to form a contextual representation supporting CFC. Thus, any potential molecular process required for the stabilization of the cellular representation for context must be activated within this narrow and behaviorally defined time window. Detection of the immediate-early gene Arc presents an ideal method to assess the activation of specific neuronal ensembles, given past studies showing the context specific expression of Arc in CA3 and CA1 subfields and the role of Arc in hippocampal long-term synaptic plasticity. Therefore, we examined the temporal dynamics of Arc induction within the hippocampus after brief context exposure to determine whether experience-dependent Arc expression could be involved in the rapid encoding of incidental context memories. We found that the duration of context exposure differentially activated Arc expression in hippocampal subfields, with CA3 showing rapid engagement within as little as 3 s of exposure. By contrast, Arc induction in CA1 required 30 s of context exposure to reach maximal levels. A parallel behavioral experiment revealed that 30 s, but not 3 s, exposure to a context resulted in strong conditioned freezing 24 h later, consistent with past studies from other laboratories. The current study is the first to examine the rapid temporal dynamics of Arc induction in hippocampus in a well-defined context memory paradigm. These studies demonstrate within 30 s of context exposure Arc is fully activated in CA3 and CA1, suggesting that the engagement of plastic processes requiring Arc function (such as long-term potentiation) occurs within the same temporal domain as that required for behavioral conditioning.

Propofol is a rapidly acting water-insoluble non-barbiturate anesthetic agent that is widely used as an intravenous sedative-hypnotic agent. Anecdotal evidence indicates that propofol may be effective at terminating intractable migraine headache. Cortical spreading depression (CSD) is believed to be the neural correlate of migraine aura and may be a trigger for migraine pain. Agents that block the induction or slow the spread of CSD may be of utility in treating migraine. Here we examined the ability of propofol hemisuccinate (PHS), a water-soluble prodrug of propofol, to affect CSD in mice. For comparison, we examinined dizocilpine, an NMDA receptor antagonist, that is well recognized to inhibit CSD. When administered 15min prior to activation of CSD by KCl application to the cortex, intraperitoneal PHS at doses of 120 and 200mg/kg decreased the number of CSD deflections without an effect on CSD amplitude, and at 200mg/kg caused a 77% reduction in CSD velocity. The minimally-effective dose of PHS (120mg/kg) did not cause sedation or motor impairment and while some animals receiving 200mg/kg did demonstrate motor impariment none exhibited loss-of-righting reflex (anesthesia). Dizocilpine produced comparable inhibition of CSD at doses of 0.5 and 2.5mg/kg. We conclude that acute PHS treatment inhibits CSD. Our results indicate that propofol, or its prodrug PHS, are worthy of further investigation as a treatment for migraine.

Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the protein phosphatases family and has a dual function in cell cycling. The function of this gene has been studied in several kinds of cancers, but its role in human hepatocellular carcinoma (HCC) remains to be elucidated. In this study, we found that CDKN3 was frequently overexpressed in both HCC cell lines and clinical samples, and this overexpression was correlated with poor tumor differentiation and advanced tumor stage. Functional studies showed that overexpression of CDKN3 could promote cell proliferation by stimulating G1-S transition but has no impact on cell apoptosis and invasion. Microarray-based co-expression analysis identified a total of 61 genes co-expressed with CDKN3, with most of them involved in cell proliferation, and BIRC5 was located at the center of CDKN3 co-expression network. These results suggest that CDKN3 acts as an oncogene in human hepatocellular carcinoma and antagonism of CDKN3 may be of interest for the treatment of HCC.

This work compares the effects of electrical stimulation of the paraventricular hypothalamic nucleus (PVN) and the raphe magnus nucleus (RMg) on the single-unit response from dorsal spinal cord neurons activated by nociceptive receptive field stimulation. We evaluated the effects of stimulating the PVN or RMg individually or simultaneously, as well as PVN stimulation after RMg electrolytic lesion. PVN or RMg stimulation suppressed the A-delta, C fiber, and postdischarge, and we demonstrated that their simultaneous stimulation increases the duration and intensity of suppressive effects. RMg lesion increased the peripheral responses, but PVN stimulation continued to be suppressive. The intrathecal administration of 20 μl of a 10⁻⁵ M solution of a specific oxytocin antagonist strongly reduced the PVN effects, and 20 μl of 10⁻⁶ M naloxone significantly reduced the RMg suppression of receptive field responses. Some spinal cord cells presented a short-latency, evoked action potential (6.8 ms and a variability of ±0.5 ms) produced by the RMg stimulation. This is interpreted as a direct postsynaptic action of the RMg on the spinal cord cells. We never found similar responses produced by the PVN, and therefore, we propose that the PVN effects are presynaptic. Finally, the immunohistochemical experiments confirmed the oxytocinergic and the vasopresinergic innervation used by the PVN projection to the RMg, and they raise the possibility that other neurotransmitters are involved. We conclude that the PVN and the RMg form part of a homeostatic analgesic mechanism acting on the same spinal cord cells to block the noxious information, but using different mechanisms. Both structures, and others, contribute to the homeostatic mechanism of endogenous analgesia.

Cellular morphogenesis, including polarized outgrowth, promotes tissue shape and function. Polarized vesicle trafficking has emerged as a fundamental mechanism by which protein and membrane can be targeted to discrete subcellular domains to promote localized protrusions. Frizzled (Fz)/planar cell polarity (PCP) signaling orchestrates cytoskeletal polarization and drives morphogenetic changes in such contexts as the vertebrate body axis and external Drosophila melanogaster tissues. Although regulation of Fz/PCP signaling via vesicle trafficking has been identified, the interplay between the vesicle trafficking machinery and downstream terminal PCP-directed processes is less established. In this paper, we show that Drosophila CK1-γ/gilgamesh (gish) regulates the PCP-associated process of trichome formation through effects on Rab11-mediated vesicle recycling. Although the core Fz/PCP proteins dictate prehair formation broadly, CK1-γ/gish restricts nucleation to a single site. Moreover, CK1-γ/gish works in parallel with the Fz/PCP effector multiple wing hairs, which restricts prehair formation along the perpendicular axis to Gish. Our findings suggest that polarized Rab11-mediated vesicle trafficking regulated by CK1-γ is required for PCP-directed processes.

Increasing interest in sublingual immunotherapy (SLIT) among practitioners and patients has resulted in numerous publications and clinical trials in recent years. With the clinical growth of SLIT, discussions of its efficacy, safety, and immunologic effects have intensified, as have comparisons to subcutaneous immunotherapy (SCIT). In the United States, SCIT has been the traditional form of immunotherapy for inhalant allergy and is the only immunotherapy method approved by the U.S. Food and Drug Administration at this time. The similarities and differences between SLIT and SCIT are often discussed, yet clinical studies directly comparing these immunotherapy methods are scarce.

In insects, P450s are responsible for the oxidative metabolism of structurally diverse endogenous and exogenous compounds including plant allelochemicals and insecticides. A novel full-length P450 cDNA, CYP6B47, was cloned from Spodoptera litura (Lepidoptera: Noctuidae). The sequence is 1718 bp in length with an ORF of 1509 bp encoding 503 amino acid residues. The phylogenetic analysis indicated that CYP6B47 belongs to CYP3 clan and second clade of CYP6Bs which contain 11 P450s from Noctuidae. Quantitative real-time PCR showed that CYP6B47 was expressed only in larvae stages and had a high level of transcription in the midgut and fat body. In addition, we cloned a 2141-bp 5'-flanking regions and presented the basal luciferase activities of promoter. We also predicted multiple putative elements for transcription factors binding in the 5'-flanking region. Interestingly, the expression of CYP6B47 significantly increased in the midgut and fat body after lead (Pb) exposure for 5 generations. Larvae tolerance to the alpha-cypermethrin (35% increased in LC(50)) and fenvalerate (52% increased in LC(50)) were improved after pre-exposure to 50 mg/kg Pb. These dates suggested that lead increased tolerance of larvae to insecticides mainly through transcriptional induction of detoxification genes including CYP6B47.

Endothelin ET(B) receptors mediate under normal physiological conditions vasorelaxation in coronary arteries. However, vasocontractile ET(B) receptors appear in coronary arteries of ischemic heart disease patients. Interestingly, organ culture of isolated coronary arteries also induces upregulation of vasocontractile ET(B) receptors. This study examines the early time course and mechanism behind upregulation of contractile ET(B) receptors in isolated rat coronary arteries during short-term organ culture.

Object processing is affected by the gist of the scene within which it is embedded. Previous ERP research has suggested that manipulating the semantic congruency between an object and the surrounding scene affects the high level (semantic) representation of that object emerging after the presentation of the scene (Ganis & Kutas, 2003). In two ERP experiments, we investigated whether there would be a similar electrophysiological response when spatial congruency of an object in a scene was manipulated while the semantic congruency remained the same. Apart from the location of the object, all other object features were congruent with the scene (e.g., in a bedroom scene, either a painting or a cat appeared on the wall). In the first experiment, participants were shown a location cue and then a scene image for 300 ms, after which an object image appeared on the cued location for 300 ms. Spatially incongruent objects elicited a stronger centro-frontal N300-N400 effect in the 275-500 ms window relative to the spatially congruent objects. We also found early ERP effects, dominant on the left hemisphere electrodes. Strikingly, LORETA analysis revealed that these activations were mainly located in the superior and middle temporal gyrus of the right hemisphere. In the second experiment, we used a paradigm similar to Mudrik, Lamy, and Deouell (2010). The scene and the object were presented together for 300 ms after the location cue. This time, we did not observe either an early or the pronounced N300-N400 effect. In contrast to Experiment 1, LORETA analysis on the N400 time-window revealed that the generators of these weak ERP effects were mainly located in the temporal lobe of the left hemisphere. Our results suggest that, when the scene is presented before the object, top-down spatial encoding processes are initiated and the right superior temporal gyrus is activated, as previously suggested (Ellison, Schindler, Pattison, & Milner, 2004). Mismatch between the actual object features and the spatially driven top-down structural and functional features could lead to the early effect, and then to the N300-N400 effect. In contrast, when the scene is not presented before the object, the spatial encoding could not happen early and strong enough to initiate spatial object-integration effects. Our results indicate that spatial information is an early and essential part in scene-object integration, and it primes structural as well as semantic features of an object.

Interaction between interleukin (IL)-5 and its receptor (IL-5R) is important for the regulation of immunity against worm infections, allergic reactions and B cell response in mammals. In this study, we identified a full-length cDNA encoding chicken IL-5R α-chain (chIL-5Rα). The deduced amino acid sequence showed 41-43% identity to mammalian homologues. It has four well-conserved cysteines and a WSXWS motif in the extracellular region, and a PPXP motif in the cytoplasmic region. Quantitative RT-PCR analysis revealed that chIL-5Rα mRNA expression was markedly high in bone marrow and relatively high in spleen and lung. Recombinant proteins of soluble chIL-5Rα and cytokines (artificially produced chIL-5 (achIL-5) and another IL-5-like molecule KK34) were expressed by 293F cells to examine the cytokine-receptor interactions. Interaction assay using a Biacore biosensor showed that chIL-5Rα has the capability to bind with monomeric achIL-5, but not with KK34. In conclusion, chicken has an IL-5Rα homologue but KK34 does not complement the IL-5/IL-5R system.

Nevirapine (NVP) is an anti-HIV drug associated with severe hepatotoxicity and skin rashes, which raises concerns about its chronic administration. There is increasing evidence that metabolic activation to reactive electrophiles capable of reacting with bionucleophiles is likely to be involved in the initiation of these toxic responses. Phase I NVP metabolism involves oxidation of the 4-methyl substituent and the formation of phenolic derivatives that are conceivably capable of undergoing further metabolic oxidation to electrophilic quinoid species prone to react with bionucleophiles. The covalent adducts thus formed might be at the genesis of toxic responses. As part of a program aimed at evaluating the possible contribution of quinoid derivatives of Phase I phenolic NVP metabolites to the toxic responses elicited by the parent drug, we have investigated the oxidation of 2-hydroxy-NVP with dipotassium nitroso-disulfonate (Frémy's salt), mimicking the one-electron oxidation involved in enzyme-mediated metabolic oxidations. We report herein the isolation and full structural characterization of a 1H-pyrrole-2,5-dione derivative as a major product, stemming from an unusual pyridine ring contraction.

Nine compounds have been isolated for the first time from Celtis africana, namely trans-N-coumaroyltyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), lauric acid (4), oleic acid (5), palmitic acid (6), lupeol (7), β-sitosterol (8) and oleanolic acid (9), respectively. Their structures have been elucidated by different spectroscopic techniques. The isolated compounds were screened for their antioxidant, anti-inflammatory and acetylcholinestrease enzyme inhibitory activities. Compounds 1-3 showed significant antioxidant and anti-inflammatory activities and weak to moderate acetylcholinestrease enzyme inhibition activity.

Sideritis scardica Griseb., Lamiaceae (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of antimicrobial infections, gastrointestinal complaints, inflammation and rheumatic disorders. This study reports a comparison between conventional (hydrodistillation HD and solvent extraction SE) and alternative (supercritical carbon dioxide SC CO₂) extraction methods regarding the qualitative and quantitative composition of the obtained extracts as analyzed by GC and GC-MS techniques and their anitimicrobial activity. Different types of extracts were tested, the essential oil EO obtained by HD, EO-CO₂ and AO-CO₂ obtained by SC CO₂ at different preasures 10 and 30 MPa, at 40 °C, respectively, and the fractions A, B, C and D obtained by successive solvent extraction (SE) A: ethanol, B: diethyl ether, C: ethyl acetate and D: n-butanol). While EO was characterized by the presence of the high percentage of oxygenated monoterpenes and sesquiterpenes (30.01 and 25.54%, respectively), the rest of the investigated samples were the most abundant in fatty acids and their esters and diterpenes (from 16.72 to 71.07% for fatty acids and their esters, and from 23.30 to 72.76%, for diterpenes). Microbial susceptibility tests revealed the strong to moderate activity of all investigated extracts against the tested microorganisms (MIC from 40 to 2,560 μg/mL). Although differences in the chemical compositions determined by GC and GC-MS analysis were established, the displayed antimicrobial activity was similar for the all investigated extracts.

Gamma-aminobutyric acid (GABA) and glutamate (GLU) are involved in nociceptive signals processing in the trigeminal system. In this study, we investigated the influence of excitatory transmission on GABA release in nerve terminals isolated from the rat trigeminal caudal nucleus (TCN).

We show that a single gene locus gives rise to two fully processed and functional miRNAs, i.e. that due to imperfect base pairing, two distinct microRNAs (miRNAs) can be produced from the fully complementary DNA strands. The antisense strand encodes miR-214, which is transcribed by its own promoter, whereas a novel miRNA, miR-3120, is co-expressed with its host gene mRNA. We also found that miR-3120 regulates important aspects of cellular function that are similar to that of its host gene, dynamin-3. miR-3120 was found to be located in neuronal cell bodies and to target Hsc70 and auxilin, and its lentivirus-mediated expression inhibited the uncoating of clathrin-coated vesicles. Finally, mirror miRNAs are likely to represent a new group of miRNAs with complex roles in coordinating gene expression.

Little is known about enzymatic quinone-quinol interconversions in the lipid membrane when compared with our knowledge of substrate transformations by globular enzymes. Here, the smallest example of a quinol dehydrogenase in nature, CymA, has been studied. CymA is a monotopic membrane tetraheme c-type cytochrome belonging to the NapC/NirT family and central to anaerobic respiration in Shewanella sp. Using protein-film electrochemistry, it is shown that vesicle-bound menaquinone-7 is not only a substrate for this enzyme but is also required as a cofactor when converting other quinones. Here, we propose that the high concentration of quinones in the membrane negates the evolutionary pressure to create a high affinity active site. However, the instability and reactivity of reaction intermediate, semiquinone, might require a cofactor that functions to minimize damaging side reactions.

Ras-like (Ral) small GTPases are regulated downstream of Ras and the noncanonical Ral guanine nucleotide exchange factor (RalGEF) effector pathway. Despite RalA and RalB sharing 82% sequence identity and utilization of shared effector proteins, their roles in normal and neoplastic cell growth have been shown to be highly distinct. Here, we determined that RalB function is regulated by protein kinase Cα (PKCα) phosphorylation. We found that RalB phosphorylation on Ser-198 in the C-terminal membrane targeting sequence resulted in enhanced RalB endomembrane accumulation and decreased RalB association with its effector, the exocyst component Sec5. Additionally, RalB phosphorylation regulated vesicular trafficking and membrane fusion by regulating v- and t-SNARE interactions. RalB phosphorylation regulated vesicular traffic of α5-integrin to the cell surface and cell attachment to fibronectin. In summary, our data suggest that phosphorylation by PKCα is critical for RalB-mediated vesicle trafficking and exocytosis.

Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133(+) human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2-Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF-VEGFR2-NRP1, which is associated with VEGFR2-NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF-VEGFR2-NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach.

Ag-implanted titanium with a nanostructured surface was prepared by hydrothermal treatment with H(2)O(2) followed by Ag plasma immersion ion implantation. Streptococcus mutans, Porphyromonas gingivalis and Candida albicans were chosen for antimicrobial tests. Genes related to microbial structure or adhesion, namely glucan-binding proteins B (GbpB), fimbria protein A (FimA), and agglutinin-like sequence4 (Als4), were examined. The osteoblast's attachment, viability, and quantitative analysis of osteogenic gene expression (Alp, Ocn, RunX2) on titanium surfaces were evaluated. Scanning electron microscopy (SEM) revealed that Ag nanoparticles of approximately 10 nm were incorporated on the nanostructured surface of titanium after Ag plasma immersion ion implantation. Trials showed that 93.99% of S. mutans, 93.57% of P. g, and 89.78% of C. albicans were killed on the Ag-implanted titanium with a nanostructured surface. Gene expressions from the three microorganisms confirmed the antimicrobial activities of the Ag-implanted titanium with a nanostructured surface. Furthermore, the adhesive images and viability assays indicated that the Ag-implanted titanium with a nanostructured surface did not impair osteoblasts. The expressions of osteoblast phenotype genes in cells grown on the Ag-implanted titanium surface were significantly increased. The results of this study suggest that the Ag-implanted titanium with a nanostructured surface displays good antimicrobial properties, reducing gene expressions of microorganisms, and excellent cell adhesion and osteogenic effects.

Monitoring of biomarkers, like urea, prostate-specific antigen (PSA), and osteopontin, is very important because they are related to kidney disease, prostate cancer, and ovarian cancer, respectively. It is well known that reverse iontophoresis can enhance transdermal extraction of small molecules, and even large molecules if reverse iontophoresis is used together with electroporation. Electroporation is the use of a high-voltage electrical pulse to create nanochannels within the stratum corneum, temporarily and reversibly. Reverse iontophoresis is the use of a small current to facilitate both charged and uncharged molecule transportation across the skin. The objectives of this in vitro study were to determine whether PSA and osteopontin are extractable transdermally and noninvasively and whether urea, PSA, and osteopontin can be extracted simultaneously by electroporation and reverse iontophoresis.