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Mutational analysis of cysteine residues of the insect odorant co-receptor (Orco) from Drosophila melanogaster reveals differential effects on agonist- and odorant-tuning receptor-dependent activation.
Turner RM, Derryberry SL, Kumar BN, Brittain T, Zwiebel LJ, Newcomb RD, Christie DL
(2014) J Biol Chem 289: 31837-45
MeSH Terms: Allosteric Site, Animals, Biotinylation, Calcium, Cysteine, DNA Mutational Analysis, Drosophila Proteins, Drosophila melanogaster, Epitopes, HEK293 Cells, Humans, Ion Channels, Kinetics, Mutagenesis, Site-Directed, Mutation, Odorants, Patch-Clamp Techniques, Protein Binding, Protein Structure, Tertiary, Receptors, Odorant, Thioglycolates, Triazoles
Show Abstract · Added February 19, 2015
Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects, and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains eight cysteine amino acid residues, six of which are highly conserved. In this study, we replaced individual cysteine residues with serine or alanine and expressed Orco mutants in Flp-In 293 T-Rex cells. Changes in intracellular Ca(2+) levels were used to determine responses to VUAA1. Replacement of two cysteines (Cys-429 and Cys-449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type DmelOrco. Kinetic modeling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the Cys mutants was confirmed by whole-cell voltage clamp electrophysiology. In contrast to the results from direct agonist activation of Orco, the two cysteine replacement mutants when co-expressed with a tuning receptor (DmelOR22a) showed an ∼10-fold decrease in potency for activation by 2-methyl hexanoate. Our work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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22 MeSH Terms
Mycobacterium tuberculosis subverts the TLR-2-MyD88 pathway to facilitate its translocation into the cytosol.
Rahman A, Sobia P, Gupta N, Kaer LV, Das G
(2014) PLoS One 9: e86886
MeSH Terms: Animals, Bacterial Translocation, Cytosol, DNA Primers, Digitonin, Macrophages, Peritoneal, Mice, Mice, Inbred C57BL, Microscopy, Confocal, Mycobacterium tuberculosis, Myeloid Differentiation Factor 88, Real-Time Polymerase Chain Reaction, Signal Transduction, Thioglycolates, Toll-Like Receptor 2
Show Abstract · Added March 20, 2014
Mycobacterium tuberculosis (M.tb) has evolved mechanisms to evade its destruction in phagolysosomes, where it successfully survives and replicates within phagocytes. Recent studies have shown that virulent strains of M.tb can translocate from the phagosome into the cytosol of dendritic cells (DC). The molecular mechanisms by which virulent M.tb strains can escape the phagosome remain unknown. Here we show that the virulent M.tb strain H37Rv, but not the vaccine strain Bacille Calmette-Guérin (BCG), escapes from the phagolysosome and enters the cytosol by interfering with the TLR-2-MyD88 signaling pathway. Using H37Rv mutants, we further demonstrate that the region of difference-1 (RD-1) locus and ESAT-6, a gene within the RD-1 locus, play an important role in the capacity of M.tb to migrate from the phagosome to the cytosol of macrophages. H37Rv, BCG, H37RvΔRD1, and H37RvΔESAT6 were able to translocate to the cytosol in macrophages derived from TLR-2- and MyD88-deficient animals, whereas only virulent H37Rv was able to enter the cytosol in macrophages from wild type mice. Therefore, signaling through the TLR-2-MyD88 pathway in macrophages plays an important role in confining M.tb within phagolysomes. Virulent strains of M.tb have evolved mechanisms to subvert this pathway, thus facilitating their translocation to the cytosol and to escape the toxic microenvironment of the phagosome or phagolysosome.
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15 MeSH Terms
A conserved aspartic acid is important for agonist (VUAA1) and odorant/tuning receptor-dependent activation of the insect odorant co-receptor (Orco).
Kumar BN, Taylor RW, Pask GM, Zwiebel LJ, Newcomb RD, Christie DL
(2013) PLoS One 8: e70218
MeSH Terms: Amino Acid Substitution, Animals, Anopheles, Drosophila Proteins, Drosophila melanogaster, Insect Proteins, Mutagenesis, Protein Structure, Tertiary, Receptors, Odorant, Thioglycolates, Triazoles
Show Abstract · Added May 27, 2014
Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco). An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca(2+) influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ~2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels.
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11 MeSH Terms
Analysis of inflammatory and lipid metabolic networks across RAW264.7 and thioglycolate-elicited macrophages.
Maurya MR, Gupta S, Li X, Fahy E, Dinasarapu AR, Sud M, Brown HA, Glass CK, Murphy RC, Russell DW, Dennis EA, Subramaniam S
(2013) J Lipid Res 54: 2525-42
MeSH Terms: Animals, Cell Line, Cytokines, Gene Expression Profiling, Inflammation, Lipid Metabolism, Lipopolysaccharides, Macrophages, Mice, Thioglycolates, Transcription, Genetic
Show Abstract · Added March 7, 2014
Studies of macrophage biology have been significantly advanced by the availability of cell lines such as RAW264.7 cells. However, it is unclear how these cell lines differ from primary macrophages such as thioglycolate-elicited peritoneal macrophages (TGEMs). We used the inflammatory stimulus Kdo2-lipid A (KLA) to stimulate RAW264.7 and TGEM cells. Temporal changes of lipid and gene expression levels were concomitantly measured and a systems-level analysis was performed on the fold-change data. Here we present a comprehensive comparison between the two cell types. Upon KLA treatment, both RAW264.7 and TGEM cells show a strong inflammatory response. TGEM (primary) cells show a more rapid and intense inflammatory response relative to RAW264.7 cells. DNA levels (fold-change relative to control) are reduced in RAW264.7 cells, correlating with greater downregulation of cell cycle genes. The transcriptional response suggests that the cholesterol de novo synthesis increases considerably in RAW264.7 cells, but 25-hydroxycholesterol increases considerably in TGEM cells. Overall, while RAW264.7 cells behave similarly to TGEM cells in some ways and can be used as a good model for inflammation- and immune function-related kinetic studies, they behave differently than TGEM cells in other aspects of lipid metabolism and phenotypes used as models for various disorders such as atherosclerosis.
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11 MeSH Terms
Blockade of insect odorant receptor currents by amiloride derivatives.
Pask GM, Bobkov YV, Corey EA, Ache BW, Zwiebel LJ
(2013) Chem Senses 38: 221-9
MeSH Terms: Amiloride, Animals, Anopheles, Cell Line, HEK293 Cells, Humans, Insect Proteins, Patch-Clamp Techniques, Receptors, Odorant, Thioglycolates, Transfection, Triazoles
Show Abstract · Added May 27, 2014
Insect odorant receptors (ORs) function as heteromeric odorant-gated ion channels consisting of a conserved coreceptor, Orco, and an odorant-sensitive tuning subunit. Although some OR modulators have been identified, an extended library of pharmacological tools is currently lacking and would aid in furthering our understanding of insect OR complexes. We now demonstrate that amiloride and several derivatives, which have been extensively used as blockers for various ion channels and transporters, also block odorant-gated currents from 2 OR complexes from the malaria vector mosquito Anopheles gambiae. In addition, both heteromeric and homomeric ORs were susceptible to amiloride blockade when activated by VUAA1, an agonist that targets the Orco channel subunit. Amiloride derivatives therefore represent a valuable class of channel blockers that can be used to investigate the pharmacological and biophysical properties of insect OR function.
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12 MeSH Terms
Structure-activity relationship of a broad-spectrum insect odorant receptor agonist.
Taylor RW, Romaine IM, Liu C, Murthi P, Jones PL, Waterson AG, Sulikowski GA, Zwiebel LJ
(2012) ACS Chem Biol 7: 1647-52
MeSH Terms: Animals, Anopheles, Behavior, Animal, HEK293 Cells, Humans, Ion Channels, Larva, Motor Activity, RNA, Small Interfering, Receptors, Odorant, Structure-Activity Relationship, Thioglycolates, Triazoles
Show Abstract · Added March 5, 2014
Agonism of insect odorant receptor (OR) cation channels may represent a new strategy for the manipulation of destructive insect olfactory-driven behaviors. We have explored the chemical space around VUAA1, the first in class agonist of the obligate OR co-receptor ion channel (Orco), and describe novel compound analogues with increased potency across insect taxa. Functional analyses reveal several of these VUAA1 structural analogues display significantly greater potency as compared to the activity of the previously described active compounds in mobility-based behavioral assays on mosquito larvae.
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13 MeSH Terms
Allosteric antagonism of insect odorant receptor ion channels.
Jones PL, Pask GM, Romaine IM, Taylor RW, Reid PR, Waterson AG, Sulikowski GA, Zwiebel LJ
(2012) PLoS One 7: e30304
MeSH Terms: Allosteric Regulation, Animals, Anopheles, Dose-Response Relationship, Drug, Evoked Potentials, Female, HEK293 Cells, Humans, Insect Proteins, Ion Channels, Molecular Structure, Odorants, Olfactory Receptor Neurons, Organic Chemicals, Phenothiazines, Receptors, Odorant, Structure-Activity Relationship, Thioglycolates, Triazoles
Show Abstract · Added March 5, 2014
BACKGROUND - At a molecular level, insects utilize members of several highly divergent and unrelated families of cell-surface chemosensory receptors for detection of volatile odorants. Most odors are detected via a family of odorant receptors (ORs), which form heteromeric complexes consisting of a well-conserved OR co-receptor (Orco) ion channel and a non-conserved tuning OR that provides coding specificity to each complex. Orco functions as a non-selective cation channel and is expressed in the majority of olfactory receptor neurons (ORNs). As the destructive behaviors of many insects are principally driven by olfaction, Orco represents a novel target for behavior-based control strategies. While many natural and synthetic odorants have been shown to agonize Orco/Or complexes, only a single direct Orco modulator, VUAA1, has been described. In an effort to identify additional Orco modulators, we have investigated the structure/activity relationships around VUAA1.
RESULTS - A search of our compound library identified several VUAA1 analogs that were selected for evaluation against HEK cells expressing Orco from the malaria vector Anopheles gambiae (AgOrco). While the majority of compounds displayed no activity, many of these analogs possess no intrinsic efficacy, but instead, act as competitive VUAA1 antagonists. Using calcium mobilization assays, patch clamp electrophysiology, and single sensillum in vivo recording, we demonstrate that one such candidate, VU0183254, is a specific allosteric modulator of OR signaling, capable of broadly inhibiting odor-mediated OR complex activation.
CONCLUSIONS - We have described and characterized the first Orco antagonist, that is capable of non-competitively inhibiting odorant-evoked activation of OR complexes, thereby providing additional insight into the structure/function of this unique family of ligand-gated ion channels. While Orco antagonists are likely to have limited utility in insect control programs, they represent important pharmacological tools that will facilitate the investigation of the molecular mechanisms underlying insect olfactory signal transduction.
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19 MeSH Terms
Prostaglandin E2 restrains macrophage maturation via E prostanoid receptor 2/protein kinase A signaling.
Zaslona Z, Serezani CH, Okunishi K, Aronoff DM, Peters-Golden M
(2012) Blood 119: 2358-67
MeSH Terms: Animals, Antigens, Differentiation, CD11b Antigen, Cell Line, Tumor, Cells, Cultured, Cyclic AMP-Dependent Protein Kinases, Dinoprostone, Female, Flow Cytometry, Gene Expression, Macrophage Colony-Stimulating Factor, Macrophages, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Misoprostol, Peritonitis, Prostaglandin Antagonists, Receptor, Macrophage Colony-Stimulating Factor, Receptors, Prostaglandin E, EP2 Subtype, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Thioglycolates, Xanthones
Show Abstract · Added May 4, 2017
Prostaglandin E(2) (PGE(2)) is a lipid mediator that acts by ligating 4 distinct G protein-coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE(2) in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE(2) receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2(-/-)) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2(-/-) mice exhibited a higher percentage of F4/80(high)/CD11b(high) cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE(2) analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2(-/-) mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE(2)/EP2/protein kinase A signaling in the suppression of macrophage maturation.
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25 MeSH Terms
Functional agonism of insect odorant receptor ion channels.
Jones PL, Pask GM, Rinker DC, Zwiebel LJ
(2011) Proc Natl Acad Sci U S A 108: 8821-5
MeSH Terms: Animals, Anopheles, Insecta, Ion Channels, Receptors, Odorant, Signal Transduction, Thioglycolates, Triazoles
Show Abstract · Added May 27, 2014
In insects, odor cues are discriminated through a divergent family of odorant receptors (ORs). A functional OR complex consists of both a conventional odorant-binding OR and a nonconventional coreceptor (Orco) that is highly conserved across insect taxa. Recent reports have characterized insect ORs as ion channels, but the precise mechanism of signaling remains unclear. We report the identification and characterization of an Orco family agonist, VUAA1, using the Anopheles gambiae coreceptor (AgOrco) and other orthologues. These studies reveal that the Orco family can form functional ion channels in the absence of an odor-binding OR, and in addition, demonstrate a first-in-class agonist to further research in insect OR signaling. In light of the extraordinary conservation and widespread expression of the Orco family, VUAA1 represents a powerful new family of compounds that can be used to disrupt the destructive behaviors of nuisance insects, agricultural pests, and disease vectors alike.
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8 MeSH Terms
Opposing roles of mGluR8 in measures of anxiety involving non-social and social challenges.
Duvoisin RM, Villasana L, Davis MJ, Winder DG, Raber J
(2011) Behav Brain Res 221: 50-4
MeSH Terms: Acetanilides, Aging, Anilides, Animals, Anxiety, Cyclohexanecarboxylic Acids, Disease Models, Animal, Dose-Response Relationship, Drug, Female, Interpersonal Relations, Male, Maze Learning, Mice, Mice, Inbred C57BL, Mice, Knockout, Receptors, Metabotropic Glutamate, Sensory Gating, Thioglycolates
Show Abstract · Added May 19, 2014
Metabotropic glutamate receptors (mGluRs) modulate glutamatergic and GABAergic neurotransmission. mGluR8, a member of group III receptors, is generally located presynaptically where it regulates neurotransmitter release. Previously we reported higher measures of anxiety in 6- and 12-month-old mGluR8(-/-) male mice than age- and sex-matched wild-type mice and that acute pharmacological stimulation with the mGluR8 agonist (S)-3,4,-dicarboxyphenylglycine (DCPG) or the Positive Allosteric Modulator (PAM) AZ12216052 reduced measures of anxiety in wild-type mice. As in humans and animals, ageing is associated with enhanced measures of anxiety following non-social and social challenges, increased understanding of these measures and how to potentially modulate them is particularly important in the elderly. Here we determined whether the effects of AZ12216052 on measures of anxiety are mediated by mGluR8 using 24-month-old mGluR8(-/-) and wild-type male mice. AZ12216052 also reduced measures of anxiety in the elevated zero maze and the acoustic startle response in mGluR8(-/-) mice. The remaining anxiolytic effects of AZ12216052 in mGluR8(-/-) mice might involve mGluR4, as the mGluR4 PAM VU 0155041 also reduced measures of anxiety in wild-type mice. In contrast, mGluR8(-/-) mice show enhanced social interaction but AZ12216052 does not affect social interaction in wild-type mice. Thus, while mGluR8 is an attractive target to modulate measures of anxiety and social interaction, the effects of AZ12216052 on measures of anxiety likely also involve receptors other than mGluR8.
Copyright © 2011 Elsevier B.V. All rights reserved.
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18 MeSH Terms