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Sample records for serum-dependently mediate gabaa

  1. Inflammatory mediators potentiate high affinity GABA(A) currents in rat dorsal root ganglion neurons.

    Science.gov (United States)

    Lee, Kwan Yeop; Gold, Michael S

    2012-06-19

    Following acute tissue injury action potentials may be initiated in afferent processes terminating in the dorsal horn of the spinal cord that are propagated back out to the periphery, a process referred to as a dorsal root reflex (DRR). The DRR is dependent on the activation of GABA(A) receptors. The prevailing hypothesis is that DRR is due to a depolarizing shift in the chloride equilibrium potential (E(Cl)) following an injury-induced activation of the Na(+)-K(+)-Cl(-)-cotransporter. Because inflammatory mediators (IM), such as prostaglandin E(2) are also released in the spinal cord following tissue injury, as well as evidence that E(Cl) is already depolarized in primary afferents, an alternative hypothesis is that an IM-induced increase in GABA(A) receptor mediated current (I(GABA)) could underlie the injury-induced increase in DRR. To test this hypothesis, we explored the impact of IM (prostaglandin E(2) (1 μM), bradykinin (10 μM), and histamine (1 μM)) on I(GABA) in dissociated rat dorsal root ganglion (DRG) neurons with standard whole cell patch clamp techniques. IM potentiated I(GABA) in a subpopulation of medium to large diameter capsaicin insensitive DRG neurons. This effect was dependent on the concentration of GABA, manifest only at low concentrations (emergence of injury-induced DRR. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. Bicarbonate Contributes to GABAA Receptor-Mediated Neuronal Excitation in Surgically-Resected Human Hypothalamic Hamartomas

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    Do-Young, Kim; Fenoglio, Kristina A.; Kerrigan, John F.; Rho, Jong M.

    2009-01-01

    SUMMARY The role of bicarbonate (HCO3-) in GABAA receptor-mediated depolarization of human hypothalamic hamartoma (HH) neurons was investigated using cellular electrophysiological and calcium imaging techniques. Activation of GABAA receptors with muscimol (30 μM) provoked neuronal excitation in over 70% of large (18-22 μM) HH neurons in HCO3- buffer. Subsequent perfusion of HCO3--free HEPES buffer produced partial suppression of muscimol-induced excitation. Additionally, 53% of large HH neurons under HCO3--free conditions exhibited reduced intracellular calcium accumulation by muscimol. These results suggest that HCO3- efflux through GABAA receptors on a subpopulation of large HH neurons may contribute to membrane depolarization and subsequent activation of L-type calcium channels. PMID:19022626

  3. Lindane blocks GABAA-mediated inhibition and modulates pyramidal cell excitability in the rat hippocampal slice.

    Science.gov (United States)

    Joy, R M; Walby, W F; Stark, L G; Albertson, T E

    1995-01-01

    An in vitro paired-pulse orthodromic stimulation technique was used to examine the effects of lindane on excitatory afferent terminals, CA1 pyramidal cells and recurrent collateral evoked inhibition in the rat hippocampal slice. This was done to establish simultaneous effects on a simple neural network and to develop procedures for more detailed analyses of the effects of lindane. Hippocampal slices 400 microns thick were perfused with oxygenated artificial cerebrospinal fluid. Electrodes were placed in the CA1 region to record extracellular population spike (PS) or excitatory postsynaptic potential (EPSP) responses to stimulation of Schaffer collateral/commissural (SC/C) fibers. Gamma-aminobutyric acid (GABA)-mediated recurrent inhibition was measured using a paired-pulse technique. Perfusion with lindane produced both time and dose dependent changes in a number of the responses measured. The most striking effect produced by lindane was the loss of GABAA-mediated recurrent collateral inhibition. This tended to occur rapidly, often before changes in EPSP or PS responses could be detected. With longer exposures to lindane, repetitive discharge of pyramidal cells developed resulting in multiple PSs to single stimuli. Lindane (50 microM) also completely reversed the effects of the injectable anesthetic, propofol, a compound known to potentiate GABAA-mediated inhibition via a direct action on the GABAA receptor-chloride channel complex. An analysis of input/output relationships at varying stimulus intensities showed that lindane increased EPSP and PS response amplitudes at any given stimulus intensity resulting in a leftward shift in the EPSP amplitude/stimulus intensity, PS amplitude/stimulus intensity and PS amplitude/EPSP amplitude relationships. This effect was most noticeable with low intensity stimuli and became progressively less so as stimulus intensities approached those yielding maximal responses. In addition lindane significantly increased paired pulse

  4. GABA(A) receptors mediate orexin-A induced stimulation of food intake.

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    Kokare, Dadasaheb M; Patole, Angad M; Carta, Anna; Chopde, Chandrabhan T; Subhedar, Nishikant K

    2006-01-01

    Although the role of orexins in sleep/wake cycle and feeding behavior is well established, underlying mechanisms have not been fully understood. An attempt has been made to investigate the role of GABA(A) receptors and their benzodiazepine site on the orexin-A induced response to feeding. Different groups of rats were food deprived overnight and next day injected intracerebroventricularly (icv) with vehicle (artificial CSF; 5 microl/rat) or orexin-A (20-50 nM/rat) and the animals were given free access to food. Cumulative food intake was measured during light phase of light/dark cycle at 1-, 2-, 4- and 6-h post-injection time points. Orexin-A (30-50 nM/rat, icv) stimulated food intake at all the time points (P GABA(A) receptor agonists muscimol (25 ng/rat, icv) and diazepam (0.5 mg/kg, ip) at subeffective doses significantly potentiated the hyperphagic effect of orexin-A (30 nM/rat, icv). However, the effect was negated by the GABA(A) receptor antagonist bicuculline (1 mg/kg, ip). Interestingly, benzodiazepine receptor antagonist flumazenil (5 ng/rat, icv), augmented the orexin-A (30 nM/rat, icv) induced hyperphagia; the effect may be attributed to the intrinsic activity of the agent. The results suggest that the hyperphagic effect of orexin-A, at least in part, is mediated by enhanced GABA(A) receptor activity.

  5. Context-Dependent Modulation of GABAAR-Mediated Tonic Currents

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    Patel, Bijal; Bright, Damian P; Mortensen, Martin

    2016-01-01

    UNLABELLED: Tonic GABA currents mediated by high-affinity extrasynaptic GABAA receptors, are increasingly recognized as important regulators of cell and neuronal network excitability. Dysfunctional GABAA receptor signaling that results in modified tonic GABA currents is associated with a number o...

  6. Epilepsy, E/I balance and GABAA receptor plasticity

    Directory of Open Access Journals (Sweden)

    Jean-Marc Fritschy

    2008-03-01

    Full Text Available GABAA receptors mediate most of the fast inhibitory transmission in the CNS. They form heteromeric complexes assembled from a large family of subunit genes. The existence of multiple GABAA receptor subtypes differing in subunit composition, localization and functional properties underlies their role for fi ne-tuning of neuronal circuits and genesis of network oscillations. The differential regulation of GABAA receptor subtypes represents a major facet of homeostatic synaptic plasticity and contributes to the excitation/inhibition (E/I balance under physiological conditions and upon pathological challenges. The purpose of this review is to discuss recent fi ndings highlighting the signifi cance of GABAA receptor heterogeneity for the concept of E/I balance and its relevance for epilepsy. Specifi cally, we address the following issues: (1 role for tonic inhibition, mediated by extrasynaptic GABAA receptors, for controlling neuronal excitability; (2 signifi cance of chloride ion transport for maintenance of the E/I balance in adult brain; and (3 molecular mechanisms underlying GABAA receptor regulation (traffi cking, posttranslational modifi cation, gene transcription that are important for homoeostatic plasticity. Finally, the relevance of these fi ndings is discussed in light of the involvement of GABAA receptors in epileptic disorders, based on recent experimental studies of temporal lobe epilepsy (TLE and absence seizures and on the identifi cation of mutations in GABAA receptor subunit genes underlying familial forms of epilepsy.

  7. GABA(A receptor-mediated acceleration of aging-associated memory decline in APP/PS1 mice and its pharmacological treatment by picrotoxin.

    Directory of Open Access Journals (Sweden)

    Yuji Yoshiike

    Full Text Available Advanced age and mutations in the genes encoding amyloid precursor protein (APP and presenilin (PS1 are two serious risk factors for Alzheimer's disease (AD. Finding common pathogenic changes originating from these risks may lead to a new therapeutic strategy. We observed a decline in memory performance and reduction in hippocampal long-term potentiation (LTP in both mature adult (9-15 months transgenic APP/PS1 mice and old (19-25 months non-transgenic (nonTg mice. By contrast, in the presence of bicuculline, a GABA(A receptor antagonist, LTP in adult APP/PS1 mice and old nonTg mice was larger than that in adult nonTg mice. The increased LTP levels in bicuculline-treated slices suggested that GABA(A receptor-mediated inhibition in adult APP/PS1 and old nonTg mice was upregulated. Assuming that enhanced inhibition of LTP mediates memory decline in APP/PS1 mice, we rescued memory deficits in adult APP/PS1 mice by treating them with another GABA(A receptor antagonist, picrotoxin (PTX, at a non-epileptic dose for 10 days. Among the saline vehicle-treated groups, substantially higher levels of synaptic proteins such as GABA(A receptor alpha1 subunit, PSD95, and NR2B were observed in APP/PS1 mice than in nonTg control mice. This difference was insignificant among PTX-treated groups, suggesting that memory decline in APP/PS1 mice may result from changes in synaptic protein levels through homeostatic mechanisms. Several independent studies reported previously in aged rodents both an increased level of GABA(A receptor alpha1 subunit and improvement of cognitive functions by long term GABA(A receptor antagonist treatment. Therefore, reduced LTP linked to enhanced GABA(A receptor-mediated inhibition may be triggered by aging and may be accelerated by familial AD-linked gene products like Abeta and mutant PS1, leading to cognitive decline that is pharmacologically treatable at least at this stage of disease progression in mice.

  8. Decrement of GABAA receptor-mediated inhibitory postsynaptic currents in dentate granule cells in epileptic hippocampus.

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    Isokawa, M

    1996-05-01

    1. Inhibitory postsynaptic currents (IPSCs) were studied in hippocampal dentate granule cells (DGCs) in the pilocarpine model and human temporal lobe epilepsy, with the use of the whole cell patch-clamp recording technique in slice preparations. 2. In the pilocarpine model, hippocampal slices were prepared from rats that were allowed to experience spontaneous seizures for 2 mo. Human hippocampal specimens were obtained from epileptic patients who underwent surgical treatment for medically intractable seizures. 3. IPSCs were generated by single perforant path stimulation and recorded at a membrane potential (Vm) of 0 mV near the reversal potential of glutamate excitatory postsynaptic currents in the voltage-clamp recording. IPSCs were pharmacologically identified as gamma-aminobutyric acid-A (GABAA) IPSCs by 10 microM bicuculline methiodide. 4. During low-frequency stimulation, IPSCs were not different in amplitude among non-seizure-experienced rat hippocampi, human nonsclerotic hippocampi, seizure-experienced rat hippocampi, and human sclerotic hippocampi. In the last two groups of DGCs, current-clamp recordings indicated the presence of prolonged excitatory postsynaptic potentials (EPSPs) mediated by the N-methyl-D-aspartate (NMDA) receptor. 5. High-frequency stimulation, administered at Vm = -30 mV to activate NMDA currents, reduced GABAA IPSC amplitude specifically in seizure-experienced rat hippocampi (t = 2.5, P < 0.03) and human sclerotic hippocampi (t = 7.7, P < 0.01). This reduction was blocked by an NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV) (50 microM). The time for GABAA IPSCs to recover to their original amplitude was also shortened by the application of APV. 6. I conclude that, when intensively activated, NMDA receptor-mediated excitatory transmission may interact with GABAergic synaptic inhibition in DGCs in seizure-experienced hippocampus to transiently reduce GABA(A) receptor-channel function. Such interactions may contribute to

  9. K-Cl Cotransporter 2-mediated Cl- Extrusion Determines Developmental Stage-dependent Impact of Propofol Anesthesia on Dendritic Spines.

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    Puskarjov, Martin; Fiumelli, Hubert; Briner, Adrian; Bodogan, Timea; Demeter, Kornel; Lacoh, Claudia-Marvine; Mavrovic, Martina; Blaesse, Peter; Kaila, Kai; Vutskits, Laszlo

    2017-05-01

    General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. The KCC2-dependent developmental increase in the efficacy of GABAA-mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

  10. Metabotropic Regulation of Extrasynaptic GABAA Receptors

    Directory of Open Access Journals (Sweden)

    William Martin Connelly

    2013-10-01

    Full Text Available A large body of work now shows the importance of GABAA receptor-mediated tonic inhibition in regulating CNS function. However, outside of pathological conditions, there is relatively little evidence that the magnitude of tonic inhibition is itself under regulation. Here we review the mechanisms by which tonic inhibition is known to be modulated, and outline the potential behavioural consequences of this modulation. Specifically, we address the ability of protein kinase A and C to phosphorylate the extrasynaptic receptors responsible for the tonic GABAA current, and how G-protein coupled receptors can regulate tonic inhibition through these effectors. We then speculate about the possible functional consequences of regulating the magnitude of the tonic GABAA current.

  11. α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth

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    Sengupta, Soma; Weeraratne, Shyamal Dilhan; Sun, Hongyu; Phallen, Jillian; Rallapalli, Sundari K.; Teider, Natalia; Kosaras, Bela; Amani, Vladimir; Pierre-Francois, Jessica; Tang, Yujie; Nguyen, Brian; Yu, Furong; Schubert, Simone; Balansay, Brianna; Mathios, Dimitris; Lechpammer, Mirna; Archer, Tenley C.; Tran, Phuoc; Reimer, Richard J.; Cook, James M.; Lim, Michael; Jensen, Frances E.; Pomeroy, Scott L.; Cho, Yoon-Jae

    2013-01-01

    Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors, and importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABR5, which encodes the α-subunit of the GABAA receptor complex, in aggressive MYC-driven, “Group 3” medulloblastomas. We hypothesized that modulation of α-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABR5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABR5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOX5 target gene expression. siRNA-mediated knockdown of HOX5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABR5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOX5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor. PMID:24196163

  12. α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth.

    Science.gov (United States)

    Sengupta, Soma; Weeraratne, Shyamal Dilhan; Sun, Hongyu; Phallen, Jillian; Rallapalli, Sundari K; Teider, Natalia; Kosaras, Bela; Amani, Vladimir; Pierre-Francois, Jessica; Tang, Yujie; Nguyen, Brian; Yu, Furong; Schubert, Simone; Balansay, Brianna; Mathios, Dimitris; Lechpammer, Mirna; Archer, Tenley C; Tran, Phuoc; Reimer, Richard J; Cook, James M; Lim, Michael; Jensen, Frances E; Pomeroy, Scott L; Cho, Yoon-Jae

    2014-04-01

    Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors and, importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABRA5, which encodes the α5-subunit of the GABAA receptor complex, in aggressive MYC-driven, "Group 3" medulloblastomas. We hypothesized that modulation of α5-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABRA5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABRA5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOXA5 target gene expression. siRNA-mediated knockdown of HOXA5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABRA5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOXA5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor.

  13. The effect of propofol on CA1 pyramidal cell excitability and GABAA-mediated inhibition in the rat hippocampal slice.

    Science.gov (United States)

    Albertson, T E; Walby, W F; Stark, L G; Joy, R M

    1996-05-24

    An in vitro paired-pulse orthodromic stimulation technique was used to examine the effects of propofol on excitatory afferent terminals, CA1 pyramidal cells and recurrent collateral evoked inhibition in the rat hippocampal slice. Hippocampal slices 400 microns thick were perfused with oxygenated artificial cerebrospinal fluid, and electrodes were placed in the CA1 region to record extracellular field population spike (PS) or excitatory postsynaptic potential (EPSP) responses to stimulation of Schaffer collateral/commissural fibers. Gamma-aminobutyric acid (GABA)-mediated recurrent inhibition was measured using a paired-pulse technique. The major effect of propofol (7-28 microM) was a dose and time dependent increase in the intensity and duration of GABA-mediated inhibition. This propofol effect could be rapidly and completely reversed by exposure to known GABAA antagonists, including picrotoxin, bicuculline and pentylenetetrazol. It was also reversed by the chloride channel antagonist, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). It was not antagonized by central (flumazenil) or peripheral (PK11195) benzodiazepine antagonists. Reversal of endogenous inhibition was also noted with the antagonists picrotoxin and pentylenetetrazol. Input/output curves constructed using stimulus propofol caused only a small enhancement of EPSPs at higher stimulus intensities but had no effect on PS amplitudes. These studies are consistent with propofol having a GABAA-chloride channel mechanism causing its effect on recurrent collateral evoked inhibition in the rat hippocampal slice.

  14. GABA-A Receptors Mediate Tonic Inhibition and Neurosteroid Sensitivity in the Brain.

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    Reddy, Doodipala Samba

    2018-01-01

    Neurosteroids like allopregnanolone (AP) are positive allosteric modulators of synaptic and extrasynaptic GABA-A receptors. AP and related neurosteroids exhibit a greater potency for δ-containing extrasynaptic receptors. The δGABA-A receptors, which are expressed extrasynaptically in the dentate gyrus and other regions, contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. Levels of endogenous neurosteroids fluctuate with ovarian cycle. Natural and synthetic neurosteroids maximally potentiate tonic inhibition in the hippocampus and provide robust protection against a variety of limbic seizures and status epilepticus. Recently, a consensus neurosteroid pharmacophore model has been proposed at extrasynaptic δGABA-A receptors based on structure-activity relationship for functional activation of tonic currents and seizure protection. Aside from anticonvulsant actions, neurosteroids have been found to be powerful anxiolytic and anesthetic agents. Neurosteroids and Zn 2+ have preferential affinity for δ-containing receptors. Thus, Zn 2+ can prevent neurosteroid activation of extrasynaptic δGABA-A receptor-mediated tonic inhibition. Recently, we demonstrated that Zn 2+ selectively inhibits extrasynaptic δGABA-A receptors and thereby fully prevents AP activation of tonic inhibition and seizure protection. We confirmed that neurosteroids exhibit greater sensitivity at extrasynaptic δGABA-A receptors. Overall, extrasynaptic GABA-A receptors are primary mediators of tonic inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurological disorders. © 2018 Elsevier Inc. All rights reserved.

  15. Increased GABA(A) inhibition of the RVLM after hindlimb unloading in rats

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    Moffitt, Julia A.; Heesch, Cheryl M.; Hasser, Eileen M.

    2002-01-01

    Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.

  16. Anxiety and Depression: Mouse Genetics and Pharmacological Approaches to the Role of GABAA Receptor Subtypes

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    Smith, Kiersten S.; Rudolph, Uwe

    2012-01-01

    GABAA receptors mediate fast synaptic inhibitory neurotransmission throughout the central nervous system. Recent work indicates a role for GABAA receptors in physiologically modulating anxiety and depression levels. In this review, we summarize research that led to the identification of the essential role of GABAA receptors in counteracting trait anxiety and depression-related behaviors, and research aimed at identifying individual GABAA receptor subtypes involved in physiological and pharmacological modulation of emotions. PMID:21810433

  17. THIP, a hypnotic and antinociceptive drug, enhances a tonic GABAA receptor mediated conductance in mouse neocortex

    DEFF Research Database (Denmark)

    Drasbek, Kim Ryun; Jensen, Kimmo

    2006-01-01

    its cellular actions in the neocortex are uncertain, we studied the effects of THIP on neurons in slices of frontoparietal neocortex of 13- to 19-day-old (P13-19) mice. Using whole-cell patch-clamp recordings, we found that the clinically relevant THIP concentration of 1 μM induced a robust tonic GABA...... suggest that THIP activates an extrasynaptic GABA(A) receptor-mediated conductance in the neocortex, which may alter the cortical network activity....

  18. Dose-dependent EEG effects of zolpidem provide evidence for GABA(A) receptor subtype selectivity in vivo.

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    Visser, S A G; Wolters, F L C; van der Graaf, P H; Peletier, L A; Danhof, M

    2003-03-01

    Zolpidem is a nonbenzodiazepine GABA(A) receptor modulator that binds in vitro with high affinity to GABA(A) receptors expressing alpha(1) subunits but with relatively low affinity to receptors expressing alpha(2), alpha(3), and alpha(5) subunits. In the present study, it was investigated whether this subtype selectivity could be detected and quantified in vivo. Three doses (1.25, 5, and 25 mg) of zolpidem were administered to rats in an intravenous infusion over 5 min. The time course of the plasma concentrations was determined in conjunction with the change in the beta-frequency range of the EEG as pharmacodynamic endpoint. The concentration-effect relationship of the three doses showed a dose-dependent maximum effect and a dose-dependent potency. The data were analyzed for one- or two-site binding using two pharmacodynamic models based on 1) the descriptive model and 2) a novel mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for GABA(A) receptor modulators that aims to separates drug- and system-specific properties, thereby allowing the estimation of in vivo affinity and efficacy. The application of two-site models significantly improved the fits compared with one-site models. Furthermore, in contrast to the descriptive model, the mechanism-based PK/PD model yielded dose-independent estimates for affinity (97 +/- 40 and 33,100 +/- 14,800 ng x ml(-1)). In conclusion, the mechanism-based PK/PD model is able to describe and explain the observed dose-dependent EEG effects of zolpidem and suggests the subtype selectivity of zolpidem in vivo.

  19. Context-Dependent Modulation of αβγ and αβγ GABAA Receptors by Penicillin: Implications for Phasic and Tonic Inhibition

    OpenAIRE

    Feng, Hua-Jun; Botzolakis, Emmanuel J.; Macdonald, Robert L.

    2008-01-01

    Penicillin, an open-channel blocker of GABAA receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABAA receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents e...

  20. Muscarinic Long-Term Enhancement of Tonic and Phasic GABAA Inhibition in Rat CA1 Pyramidal Neurons

    Science.gov (United States)

    Domínguez, Soledad; Fernández de Sevilla, David; Buño, Washington

    2016-01-01

    Acetylcholine (ACh) regulates network operation in the hippocampus by controlling excitation and inhibition in rat CA1 pyramidal neurons (PCs), the latter through gamma-aminobutyric acid type-A receptors (GABAARs). Although, the enhancing effects of ACh on GABAARs have been reported (Dominguez et al., 2014, 2015), its role in regulating tonic GABAA inhibition has not been explored in depth. Therefore, we aimed at determining the effects of the activation of ACh receptors on responses mediated by synaptic and extrasynaptic GABAARs. Here, we show that under blockade of ionotropic glutamate receptors ACh, acting through muscarinic type 1 receptors, paired with post-synaptic depolarization induced a long-term enhancement of tonic GABAA currents (tGABAA) and puff-evoked GABAA currents (pGABAA). ACh combined with depolarization also potentiated IPSCs (i.e., phasic inhibition) in the same PCs, without signs of interactions of synaptic responses with pGABAA and tGABAA, suggesting the contribution of two different GABAA receptor pools. The long-term enhancement of GABAA currents and IPSCs reduced the excitability of PCs, possibly regulating plasticity and learning in behaving animals. PMID:27833531

  1. Neonatal blockade of GABA-A receptors alters behavioral and physiological phenotypes in adult mice.

    Science.gov (United States)

    Salari, Ali-Akbar; Amani, Mohammad

    2017-04-01

    Gamma-aminobutyric acid (GABA) plays an inhibitory role in the mature brain, and has a complex and bidirectional effect in different parts of the immature brain which affects proliferation, migration and differentiation of neurons during development. There is also increasing evidence suggesting that activation or blockade of the GABA-A receptors during early life can induce brain and behavioral abnormalities in adulthood. We investigated whether neonatal blockade of the GABA-A receptors by bicuculline can alter anxiety- and depression-like behaviors, body weight, food intake, corticosterone and testosterone levels in adult mice (postnatal days 80-95). To this end, neonatal mice were treated with either DMSO or bicuculline (70, 150 and 300μg/kg) during postnatal days 7, 9 and 11. When grown to adulthood, mice were exposed to behavioral tests to measure anxiety- (elevated plus-maze and light-dark box) and depression-like behaviors (tail suspension test and forced swim test). Stress-induced serum corticosterone and testosterone levels, body weight and food intake were also evaluated. Neonatal bicuculline exposure at dose of 300μg/kg decreased anxiety-like behavior, stress-induced corticosterone levels and increased testosterone levels, body weight and food intake, without significantly influencing depression-like behavior in adult male mice. However, no significant changes in these parameters were observed in adult females. These findings suggest that neonatal blockade of GABA-A receptors affects anxiety-like behavior, physiological and hormonal parameters in a sex-dependent manner in mice. Taken together, these data corroborate the concept that GABA-A receptors during early life have an important role in programming neurobehavioral phenotypes in adulthood. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

  2. Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice

    Science.gov (United States)

    Smith, Kiersten S.; Engin, Elif; Meloni, Edward G.; Rudolph, Uwe

    2012-01-01

    GABAA receptor modulating drugs such as benzodiazepines (BZs) have been used to treat anxiety disorders for over five decades. In order to determine whether the same or different GABAA receptor subtypes are necessary for the anxiolytic-like action of BZs in unconditioned anxiety and conditioned fear models, we investigated the role of different GABAA receptor subtypes by challenging wild type, α1(H101R), α2(H101R) and α3(H126R) mice bred on the C57BL/6J background with diazepam or chlordiazepoxide in the elevated plus maze and the fear-potentiated startle paradigms. Both drugs significantly increased open arm exploration in the elevated plus maze in wild type, α1(H101R) and α3(H126R), but this effect was abolished in α2(H101R) mice; these were expected results based on previous published results. In contrast, while administration of diazepam and chlordiazepoxide significantly attenuated fear-potentiated startle (FPS) in wild type mice and α3(H126R) mice, the fear-reducing effects of these drugs were absent in both α1(H101R) and α2(H101R) point mutants, indicating that both α1- and α2-containing GABAA receptors are necessary for BZs to exert their effects on conditioned fear responses.. Our findings illustrate both an overlap and a divergence between the GABAA receptor subtype requirements for the impact of BZs, specifically that both α1- and α2-containing GABAA receptors are necessary for BZs to reduce conditioned fear whereas only α2-containing GABAA receptors are needed for BZ-induced anxiolysis in unconditioned tests of anxiety. This raises the possibility that GABAergic pharmacological interventions for specific anxiety disorders can be differentially tailored. PMID:22465203

  3. The role of GABA-A and mitochondrial diazepam-binding inhibitor receptors on the effects of neurosteroids on food intake in mice.

    Science.gov (United States)

    Reddy, D S; Kulkarni, S K

    1998-06-01

    The present studies were undertaken to investigate the neuroactive steroidal modulation of feeding behavior and possible involvement of gamma-aminobutyric acid type-A (GABA-A) and mitochondrial diazepam binding inhibitor (DBI) receptors (MDR) in food-deprived male mice. Allopregnanolone (0.5-2 mg/kg), a neurosteroid, progesterone (1-10 mg/kg), a neurosteroid precursor, and 4'-chlordiazepam (0.25-1 mg/kg), a specific high affinity MDR agonist, produced a dose-dependent hyperphagic effects. In contrast, neurosteroids pregnenolone sulfate (PS) (1-10 mg/kg) and dehydroepiandrosterone sulfate (DHEAS) (1-10 mg/kg) produced a hypophagic effect, in a dose-dependent manner. The allopregnanolone-, progesterone- and 4'-chlordiazepam-induced hyperphagic effect was blocked by picrotoxin (1 mg/kg), a GABA-A chloride channel antagonist, but not by flumazenil (2 mg/kg), a benzodiazepine (BZD) antagonist. The 4'-chlordiazepam-induced hyperphagic effect was prevented by pretreatment with PK11195 (2 mg/kg), a selective partial MDR antagonist. The hypophagic effect of DHEAS (10 mg/kg) was reversed by dizocilpine (10 microg/kg), an NMDA receptor antagonist, but resistant to muscimol (0.1 mg/kg), a selective GABA-A receptor agonist. In contrast, the PS (10 mg/kg)-induced hypophagic response was resistant to dizocilpine, but sensitive to muscimol (0.1 mg/kg). Both the sulfated neurosteroids PS and DHEAS also reversed the hyperphagic effect of allopregnanolone. In addition, the BZD agonist triazolam (0.05-0.25 mg/kg) also produced a flumazenil- and picrotoxin-sensitive hyperphagic effects, thereby suggesting the changes in feeding behavior by neurosteroids represent GABA-A receptor mediated hyperphagic action. Although the possible antistress or anxiolytic actions of neurosteroids may confound the hyperphagia, behavioral effects observed were specific to food because the mice were adopted to the test environment and diet, and of a possible variation between various neurosteroids in the

  4. GABA regulates the rat hypothalamic-pituitary-adrenocortical axis via different GABA-A receptor alpha-subtypes

    DEFF Research Database (Denmark)

    Mikkelsen, Jens D; Bundzikova, Jana; Larsen, Marianne Hald

    2008-01-01

    dependent on the composition of the GABA-A receptor subunits through which they act. We show here that positive modulators of alpha(1)-subtype containing GABA-A receptors with zolpidem (10 mg/kg) increase HPA activity in terms of increase in plasma corticosterone and induction of Fos in the PVN, whereas...... after positive modulation of GABA-A receptors composed of alpha(1)-subunit(s) affects a selective afferent system than the PVN, which is distinct from another afferent system(s) activated by non alpha(1)-containing GABA-A receptors....

  5. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    DEFF Research Database (Denmark)

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis

    2012-01-01

    δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep......δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA......(A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 μM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 μM), while interneurons showed a potentiation of 2.6-fold......-free environment using Ca²⁺ imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition...

  6. Astrocytes Modulate a Postsynaptic NMDA–GABAA-Receptor Crosstalk in Hypothalamic Neurosecretory Neurons

    Science.gov (United States)

    Potapenko, Evgeniy S.; Biancardi, Vinicia C.; Zhou, Yiqiang

    2013-01-01

    A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABAAergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABAA receptors (GABAARs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABAARs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR–GABAAR crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate–GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate–GABA interactions in MNCs, which involves crosstalk between NMDA and GABAA postsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABAARs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure. PMID:23303942

  7. Basolateral amygdala GABA-A receptors mediate stress-induced memory retrieval impairment in rats.

    Science.gov (United States)

    Sardari, Maryam; Rezayof, Ameneh; Khodagholi, Fariba; Zarrindast, Mohammad-Reza

    2014-04-01

    The present study was designed to investigate the involvement of GABA-A receptors of the basolateral amygdala (BLA) in the impairing effect of acute stress on memory retrieval. The BLAs of adult male Wistar rats were bilaterally cannulated and memory retrieval was measured in a step-through type passive avoidance apparatus. Acute stress was evoked by placing the animals on an elevated platform for 10, 20 and 30 min. The results indicated that exposure to 20 and 30 min stress, but not 10 min, before memory retrieval testing (pre-test exposure to stress) decreased the step-through latency, indicating stress-induced memory retrieval impairment. Intra-BLA microinjection of a GABA-A receptor agonist, muscimol (0.005-0.02 μg/rat), 5 min before exposure to an ineffective stress (10 min exposure to stress) induced memory retrieval impairment. It is important to note that pre-test intra-BLA microinjection of the same doses of muscimol had no effect on memory retrieval in the rats unexposed to 10 min stress. The blockade of GABA-A receptors of the BLA by injecting an antagonist, bicuculline (0.4-0.5 μg/rat), 5 min before 20 min exposure to stress, prevented stress-induced memory retrieval. Pre-test intra-BLA microinjection of the same doses of bicuculline (0.4-0.5 μg/rat) in rats unexposed to 20 min stress had no effect on memory retrieval. In addition, pre-treatment with bicuculline (0.1-0.4 μg/rat, intra-BLA) reversed muscimol (0.02 μg/rat, intra-BLA)-induced potentiation on the effect of stress in passive avoidance learning. It can be concluded that pre-test exposure to stress can induce memory retrieval impairment and the BLA GABA-A receptors may be involved in stress-induced memory retrieval impairment.

  8. Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

    Directory of Open Access Journals (Sweden)

    Sandeep eKumar

    2012-04-01

    Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

  9. Distinct roles of synaptic and extrasynaptic GABAA receptors in striatal inhibition dynamics

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    Ruixi eLuo

    2013-11-01

    Full Text Available Striatonigral and striatopallidal projecting medium spiny neurons (MSNs express dopamine D1 (D1+ and D2 receptors (D2+, respectively. Both classes receive extensive GABAergic input via expression of synaptic, perisynaptic and extrasynaptic GABAA receptors. The activation patterns of different presynaptic GABAergic neurons produce transient and sustained GABAA receptor-mediated conductance that fulfill distinct physiological roles. We performed single and dual whole cell recordings from striatal neurons in mice expressing fluorescent proteins in interneurons and MSNs. We report specific inhibitory dynamics produced by distinct activation patterns of presynaptic GABAergic neurons as source of synaptic, perisynaptic and extrasynaptic inhibition. Synaptic GABAA receptors in MSNs contain the α2, γ2 and a β subunit. In addition, there is evidence for the developmental increase of the α1 subunit that contributes to faster inhibitory postsynaptic current (IPSC. Tonic GABAergic currents in MSNs from adult mice are carried by extrasynaptic receptors containing the α4 and δ subunit, while in younger mice this current is mediated by receptors that contain the α5 subunit. Both forms of tonic currents are differentially expressed in D1+ and D2+ MSNs. This study extends these findings by relating presynaptic activation with pharmacological analysis of inhibitory conductance in mice where the β3 subunit is conditionally removed in fluorescently labeled D2+ MSNs and in mice with global deletion of the δ subunit. Our results show that responses to low doses of gaboxadol (2μM, a GABAA receptor agonist with preference to δ subunit, are abolished in the δ but not the β3 subunit knock out mice. This suggests that the β3 subunit is not a component of the adult extrasynaptic receptor pool, in contrast to what has been shown for tonic current in young mice. Deletion of the β3 subunit from D2+ MSNs however, removed slow spontaneous IPSCs, implicating its

  10. Increased GABA-A receptor binding and reduced connectivity at the motor cortex in children with hemiplegic cerebral palsy: a multimodal investigation using 18F-fluoroflumazenil PET, immunohistochemistry, and MR imaging.

    Science.gov (United States)

    Park, Hae-Jeong; Kim, Chul Hoon; Park, Eun Sook; Park, Bumhee; Oh, So Ra; Oh, Maeng-Keun; Park, Chang Il; Lee, Jong Doo

    2013-08-01

    γ-aminobutyric acid (GABA)-A receptor-mediated neural transmission is important to promote practice-dependent plasticity after brain injury. This study investigated alterations in GABA-A receptor binding and functional and anatomic connectivity within the motor cortex in children with cerebral palsy (CP). We conducted (18)F-fluoroflumazenil PET on children with hemiplegic CP to investigate whether in vivo GABA-A receptor binding is altered in the ipsilateral or contralateral hemisphere of the lesion site. To evaluate changes in the GABA-A receptor subunit after prenatal brain injury, we performed GABA-A receptor immunohistochemistry using rat pups with a diffuse hypoxic ischemic insult. We also performed diffusion tensor MR imaging and resting-state functional MR imaging on the same children with hemiplegic CP to investigate alterations in anatomic and functional connectivity at the motor cortex with increased GABA-A receptor binding. In children with hemiplegic CP, the (18)F-fluoroflumazenil binding potential was increased within the ipsilateral motor cortex. GABA-A receptors with the α1 subunit were highly expressed exclusively within cortical layers III, IV, and VI of the motor cortex in rat pups. The motor cortex with increased GABA-A receptor binding in children with hemiplegic CP had reduced thalamocortical and corticocortical connectivity, which might be linked to increased GABA-A receptor distribution in cortical layers in rats. Increased expression of the GABA-A receptor α1 subunit within the ipsilateral motor cortex may be an important adaptive mechanism after prenatal brain injury in children with CP but may be associated with improper functional connectivity after birth and have adverse effects on the development of motor plasticity.

  11. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Singaravelu, Ragunath [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Lyn, Rodney K. [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Srinivasan, Prashanth [National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Delcorde, Julie [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Steenbergen, Rineke H.; Tyrrell, D. Lorne [Department of Medical Microbiology and Immunology, University of Alberta (Canada); Li Ka Shing Institute of Virology, Katz Centre for Pharmacy and Health Research, Edmonton, Alberta T6G 2S2 (Canada); Pezacki, John P., E-mail: John.Pezacki@nrc-cnrc.gc.ca [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada)

    2013-11-15

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.

  12. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    International Nuclear Information System (INIS)

    Singaravelu, Ragunath; Lyn, Rodney K.; Srinivasan, Prashanth; Delcorde, Julie; Steenbergen, Rineke H.; Tyrrell, D. Lorne; Pezacki, John P.

    2013-01-01

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway

  13. Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons.

    Science.gov (United States)

    Bohnsack, John Peyton; Carlson, Stephen L; Morrow, A Leslie

    2016-06-01

    The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  15. The GLP-1 Receptor Agonist Exendin-4 and Diazepam Differentially Regulate GABAA Receptor-Mediated Tonic Currents in Rat Hippocampal CA3 Pyramidal Neurons.

    Directory of Open Access Journals (Sweden)

    Sergiy V Korol

    Full Text Available Glucagon-like peptide-1 (GLP-1 is a metabolic hormone that is secreted in a glucose-dependent manner and enhances insulin secretion. GLP-1 receptors are also found in the brain where their signalling affects neuronal activity. We have previously shown that the GLP-1 receptor agonists, GLP-1 and exendin-4 enhanced GABA-activated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. The hippocampus is the centre for memory and learning and is important for cognition. Here we examined if exendin-4 similarly enhanced the GABA-activated currents in the presence of the benzodiazepine diazepam. In whole-cell recordings in rat brain slices, diazepam (1 μM, an allosteric positive modulator of GABAA receptors, alone enhanced the spontaneous inhibitory postsynaptic current (sIPSC amplitude and frequency by a factor of 1.3 and 1.6, respectively, and doubled the tonic GABAA current normally recorded in the CA3 pyramidal cells. Importantly, in the presence of exendin-4 (10 nM plus diazepam (1 μM, only the tonic but not the sIPSC currents transiently increased as compared to currents recorded in the presence of diazepam alone. The results suggest that exendin-4 potentiates a subpopulation of extrasynaptic GABAA receptors in the CA3 pyramidal neurons.

  16. Altered organization of GABAA receptor mRNA expression in the depressed suicide brain

    Directory of Open Access Journals (Sweden)

    Michael O Poulter

    2010-03-01

    Full Text Available Inter-relationships ordinarily exist between mRNA expression of GABA-A subunits in the frontopolar cortex (FPC of individuals that had died suddenly from causes other than suicide. However, these correlations were largely absent in persons that had died by suicide. In the present investigation, these findings were extended by examining GABA-A receptor expression patterns (of controls and depressed individuals that died by suicide in the orbital frontal cortex (OFC, hippocampus, amygdala. locus coeruleus (LC,and paraventricular nucleus (PVN, all of which have been implicated in either depression, anxiety or stress responsivity. Results Using QPCR analysis, we found that in controls the inter-relations between GABA-A subunits varied across brain regions, being high in the hippocampus and amygdala, intermediate in the LC, and low in the OFC and PVN. The GABA-A subunit inter-relations were markedly different in persons that died by suicide, being reduced in hippocampus and amygdala, stable in the LC, but more coordinated in the OFC and to some extent in the PVN. Conclusions It seems that altered brain region-specific inhibitory signaling, stemming from altered GABA-A subunit coordination, are associated with depression/suicide. Although, it is unknown whether GABA-A subunit re-organization was specifically tied to depression, suicide, or the accompanying distress, these data show that the co-ordinate expression of this transcriptome does vary depending on brain region and is plastic.

  17. Dynamic differentiation of GABAA-sensitive influences on orientation selectivity of complex cells in the cat striate cortex.

    Science.gov (United States)

    Pfleger, B; Bonds, A B

    1995-01-01

    The influence of GABAA receptors on orientation selectivity of cat complex cells was tested by iontophoresis of the GABAA receptor blockers bicuculline and N-methyl-bicuculline while stimulating with drifting sinusoidal gratings. Reduction of orientation tuning was markedly less than reported in previous studies that used drifting bars as visual stimuli. Only 3/31 cells lost orientation selectivity, with an average increase in bandwidth of 33%, as opposed to half the cells losing selectivity and a bandwidth increase for the remainder of 47% as reported previously. Infusion of GABAA blockers revealed a prominent stimulus onset transient response, lasting about 120 ms, that showed a broadening of orientation selectivity comparable to that found using drifting bars under similar circumstances. We believe that drifting gratings emphasize a steady-state response component that retains, in the presence of GABAA blockers, significant orientation selectivity. Because the onset transient is initially unselective for orientation, we suggest that the steady-state, orientation-selective response component develops from an alternate inhibitory mechanism, possibly mediated by GABAB receptors.

  18. The GABAA receptor agonist THIP is neuroprotective in organotypic hippocampal slice cultures

    DEFF Research Database (Denmark)

    Kristensen, Bjarne Winther; Noraberg, Jens; Zimmer, Jens

    2003-01-01

    The potential neuroprotective effects of the GABA(A) receptor agonists THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and muscimol, and the selective GluR5 kainate receptor agonist ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid), which activates GABAergic interneu......The potential neuroprotective effects of the GABA(A) receptor agonists THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and muscimol, and the selective GluR5 kainate receptor agonist ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid), which activates GABAergic...... interneurons, were examined in hippocampal slice cultures exposed to N-methyl-D-aspartate (NMDA). The NMDA-induced excitotoxicity was quantified by densitometric measurements of propidium iodide (PI) uptake. THIP (100-1000 microM) was neuroprotective in slice cultures co-exposed to NMDA (10 microM) for 48 h......, while muscimol (100-1000 microM) and ATPA (1-3 microM) were without effect. The results demonstrate that direct GABA(A) agonism can mediate neuroprotection in the hippocampus in vitro as previously suggested in vivo....

  19. Vibrio cholerae interactions with Mytilus galloprovincialis hemocytes mediated by serum components.

    Directory of Open Access Journals (Sweden)

    Laura eCanesi

    2013-12-01

    Full Text Available Edible bivalves (e.g., mussels, oysters can accumulate large amount of bacteria in their tissues and act as passive carriers of pathogens to humans. Bacterial persistence inside bivalves depends, at least in part, on hemolymph anti-bacterial activity that is exerted by both serum soluble factors and phagocytic cells (i.e., the hemocytes. It was previously shown that Mytilus galloprovincialis hemolymph serum contains opsonins that mediate D-mannose-sensitive interactions between hemocytes and V. cholerae O1 El Tor bacteria that carry the Mannose–Sensitive Hemagglutinin (MSHA. These opsonins enhance phagocytosis and killing of vibrios by facilitating their binding to hemocytes. Since V. cholerae strains not carrying the MSHA ligand (O1 classical, non O1/O139 are present in coastal water and can be entrapped by mussels, we studied whether in mussel serum, in addition to opsonins directed towards MSHA, other components can mediate opsonization of these bacteria. By comparing interactions of O1 classical and non O1/O139 strains with hemocytes in ASW and serum, it was found that M. galloprovincialis serum contains components that increase by at approximately two fold their adhesion to, association with and killing by hemocytes. Experiments conducted with high and low molecular mass fractions obtained by serum ultrafiltration indicated that these compounds have molecular mass higher than 5000 Da. Serum exposure to high temperature (80°C abolished its opsonizing capability suggesting that the involved serum active components are of protein nature. Further studies are needed to define the chemical properties and specificity of both the involved bacterial ligands and hemolymph opsonins. This information will be central not only to better understand V. cholerae ecology, but also to improve current bivalve depuration practices and properly protect human health.

  20. Studying cerebellar circuits by remote control of selected neuronal types with GABA-A receptors

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    William Wisden

    2009-12-01

    Full Text Available Although GABA-A receptor-mediated inhibition of cerebellar Purkinje cells by molecular layer interneurons (MLIs has been studied intensely on the cellular level, it has remained unclear how this inhibition regulates cerebellum-dependent behaviour. We have implemented two complementary approaches to investigate the function of the MLI-Purkinje cell synapse on the behavioral level. In the first approach we permanently disrupted inhibitory fast synaptic transmission at the synapse by genetically removing the postsynaptic GABA-A receptors from Purkinje cells (PC-Δγ2 mice. We found that chronic disruption of the MLI-Purkinje cell synapse strongly impaired cerebellar learning of the vestibular occular reflex (VOR, presumably by disrupting the temporal patterns of Purkinje cell activity. However, in PC-Δγ2 mice the baseline VOR reflex was only mildly affected; indeed PC-Δγ2 mice showed no ataxia or gait abnormalities suggesting that MLI control of Purkinje cell activity is either not involved in ongoing motor tasks or that the system has found a way to compensate for its loss. To investigate the latter possibility we have developed an alternative genetic technique; we made the MLI-Purkinje cell synapse selectively sensitive to rapid manipulation with the GABAA receptor modulator zolpidem (PC-γ2-swap mice. Minutes after intraperitoneal zolpidem injection, these PC-γ2-swap mice developed severe motor abnormalities, revealing a substantial contribution of the MLI-Purkinje cell synapse to real time motor control. The cell-type selective permanent knockout of synaptic GABAergic input, and the fast reversible modulation of GABAergic input at the same synapse illustrate how pursuing both strategies gives a fuller view.

  1. Salivary and serum inflammatory mediators among pre-conception women with periodontal disease.

    Science.gov (United States)

    Jiang, Hong; Zhang, Yiming; Xiong, Xu; Harville, Emily W; O, Karmin; Qian, Xu

    2016-12-15

    There have been inconsistent conclusions regarding the levels of inflammatory mediators in saliva and serum among people with or without periodontal disease. Although pre-conception has been put forward as the optimal time for the periodontal treatment in order to improving pregnancy outcomes, few studies have been conducted to examine inflammatory mediators in saliva and serum among pre-conception women. Pre-conception women were recruited between January 2012 and December 2014. Women were provided with an oral health examination to detect periodontal disease. Salivary and serum samples were collected at the same of examination. Inflammatory mediators includinginterleukin-1 beta (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α) and beta-glucuronidase (β-glucuronidase) were tested and analyzed among women with overall periodontal disease (n = 442) or moderate/severe periodontal disease (n = 247). Results were compared to that in women with a healthy periodontium (n = 91). Significantly increased concentrations of inflammatory mediators of IL-1β, IL-6, TNF-α and β-glucuronidase in saliva and IL-1β, β-glucuronidase and TNF-α in serum were found among pre-conception women with moderate/severe periodontal disease, compared with women without periodontal disease. Significantly increased levels were also found in all the above saliva inflammatory mediators and in serum IL-1β and TNF-α among women with overall periodontal disease. The levels of all inflammatory mediators in saliva and almost all inflammatory mediators except IL-6 in serum significantly increased with severity of periodontal disease. Periodontal disease is highly associated with the elevated levels of inflammatory mediators in saliva and some mediators in serum among pre-conception women.

  2. Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds.

    Science.gov (United States)

    Nothdurfter, Caroline; Tanasic, Sascha; Di Benedetto, Barbara; Uhr, Manfred; Wagner, Eva-Maria; Gilling, Kate E; Parsons, Chris G; Rein, Theo; Holsboer, Florian; Rupprecht, Rainer; Rammes, Gerhard

    2013-07-01

    Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

  3. GABA-independent GABAA Receptor Openings Maintain Tonic Currents

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    Wlodarczyk, Agnieszka I.; Sylantyev, Sergiy; Herd, Murray B.; Kersanté, Flavie; Lambert, Jeremy J.; Rusakov, Dmitri A.; Linthorst, Astrid C.E.; Semyanov, Alexey; Belelli, Delia; Pavlov, Ivan; Walker, Matthew C.

    2013-01-01

    Activation of GABAA receptors (GABAARs) produces two forms of inhibition: ‘phasic’ inhibition generated by the rapid, transient activation of synaptic GABAARs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of peri- or extrasynaptic GABAARs which can detect extracellular GABA. Such tonic GABAAR-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex-vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABAA receptor openings. This tonic GABAAR conductance is resistant to the competitive GABAAR antagonist SR95531, which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker picrotoxin. When slices are perfused with 200 nM GABA, a concentration that is comparable to cerebrospinal fluid concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABAARs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations. PMID:23447601

  4. Changes in the sensitivity of GABAA current rundown to drug treatments in a model of temporal lobe epilepsy

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    Pierangelo eCifelli

    2013-07-01

    Full Text Available The pharmacological treatment of mesial temporal lobe epilepsy (mTLE, the most common epileptic syndrome in adults, is still unsatisfactory, as one third of the patients are or become refractory to antiepileptic agents. Refractoriness may depend upon drug-induced alterations, but the disease per se may also undergo a progressive evolution that affects the sensitivity to drugs. mTLE has been shown to be associated with a dysfunction of the inhibitory signaling mediated by GABAA receptors. In particular, the repetitive activation of GABAA receptors produces a use-dependent decrease (rundown of the evoked currents (IGABA, which is markedly enhanced in the hippocampus and cortex of drug-resistant mTLE patients. This phenomenon has been also observed in the pilocarpine model, where the increased IGABA rundown is observed in the hippocampus at the time of the first spontaneous seizure, then extends to the cortex and remains constant in the chronic phase of the disease. Here, we examined the sensitivity of IGABA to pharmacological modulation. We focused on the antiepileptic agent levetiracetam and on the neurotrophin BDNF, which were previously reported to attenuate mTLE-induced increased rundown in the chronic human tissue. In the pilocarpine model, BDNF displayed a paramount effect, decreasing rundown in the hippocampus at the time of the first seizure, as well as in the hippocampus and cortex in the chronic period. In contrast, levetiracetam did not affect rundown in the hippocampus, but attenuated it in the cortex. Interestingly, this effect of levetiracetam was also observed on the still unaltered rundown observed in the cortex at the time of the first spontaneous seizure. These data suggest that the sensitivity of GABAA receptors to pharmacological interventions undergoes changes during the natural history of mTLE, implicating that the site of seizure initiation and the timing of treatment may highly affect the therapeutic outcome.

  5. Positive allosteric modulation of GABA-A receptors reduces capsaicin-induced primary and secondary hypersensitivity in rats

    DEFF Research Database (Denmark)

    Hansen, Rikke Rie; Erichsen, Helle K; Brown, David T

    2012-01-01

    GABA-A receptor positive allosteric modulators (PAMs) mediate robust analgesia in animal models of pathological pain, in part via enhancing injury-induced loss of GABA-A-α2 and -α3 receptor function within the spinal cord. As yet, a lack of clinically suitable tool compounds has prevented this co...

  6. Photo-antagonism of the GABAA receptor.

    Science.gov (United States)

    Mortensen, Martin; Iqbal, Favaad; Pandurangan, Arun P; Hannan, Saad; Huckvale, Rosemary; Topf, Maya; Baker, James R; Smart, Trevor G

    2014-07-29

    Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation.

  7. GABAA receptor drugs and neuronal plasticity in reward and aversion: focus on the ventral tegmental area

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    Elena eVashchinkina

    2014-11-01

    Full Text Available GABAA receptors are the main fast inhibitory neurotransmitter receptors in the mammalian brain, and targets for many clinically important drugs widely used in the treatment of anxiety disorders, insomnia and in anesthesia. Nonetheless, there are significant risks associated with the long-term use of these drugs particularly related to development of tolerance and addiction. Addictive mechanisms of GABAA receptor drugs are poorly known, but recent findings suggest that those drugs may induce aberrant neuroadaptations in the brain reward circuitry. Recently, benzodiazepines, acting on synaptic GABAA receptors, and modulators of extrasynaptic GABAA receptors (THIP and neurosteroids have been found to induce plasticity in the ventral tegmental area (VTA dopamine neurons and their main target projections. Furthermore, depending whether synaptic or extrasynaptic GABAA receptor populations are activated, the behavioral outcome of repeated administration seems to correlate with rewarding or aversive behavioral responses, respectively. The VTA dopamine neurons project to forebrain centers such as the nucleus accumbens and medial prefrontal cortex, and receive afferent projections from these brain regions and especially from the extended amygdala and lateral habenula, forming the major part of the reward and aversion circuitry. Both synaptic and extrasynaptic GABAA drugs inhibit the VTA GABAergic interneurons, thus activating the VTA DA neurons by disinhibition and this way inducing glutamatergic synaptic plasticity. However, the GABAA drugs failed to alter synaptic spine numbers as studied from Golgi-Cox-stained VTA dendrites. Since the GABAergic drugs are known to depress the brain metabolism and gene expression, their likely way of inducing neuroplasticity in mature neurons is by disinhibiting the principal neurons, which remains to be rigorously tested for a number of clinically important anxiolytics, sedatives and anesthetics in different parts of

  8. Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator

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    Sean C. Piantadosi

    2016-11-01

    Full Text Available Rationale: Current first-line treatments for stress-related disorders such as Major Depressive Disorder (MDD act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted α5-PAM, a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS and treated with α5-PAM acutely (30 minutes prior to assessing behavior or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as behavioral emotionality across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusions: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

  9. [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors.

    Science.gov (United States)

    Yagle, Monica A; Martin, Michael W; de Fiebre, Christopher M; de Fiebre, NancyEllen C; Drewe, John A; Dillon, Glenn H

    2003-12-01

    Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.

  10. Waking action of ursodeoxycholic acid (UDCA involves histamine and GABAA receptor block.

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    Yevgenij Yanovsky

    Full Text Available Since ancient times ursodeoxycholic acid (UDCA, a constituent of bile, is used against gallstone formation and cholestasis. A neuroprotective action of UDCA was demonstrated recently in models of Alzheimer's disease and retinal degeneration. The mechanisms of UDCA action in the nervous system are poorly understood. We show now that UDCA promotes wakefulness during the active period of the day, lacking this activity in histamine-deficient mice. In cultured hypothalamic neurons UDCA did not affect firing rate but synchronized the firing, an effect abolished by the GABA(AR antagonist gabazine. In histaminergic neurons recorded in slices UDCA reduced amplitude and duration of spontaneous and evoked IPSCs. In acutely isolated histaminergic neurons UDCA inhibited GABA-evoked currents and sIPSCs starting at 10 µM (IC(50 = 70 µM and did not affect NMDA- and AMPA-receptor mediated currents at 100 µM. Recombinant GABA(A receptors composed of α1, β1-3 and γ2L subunits expressed in HEK293 cells displayed a sensitivity to UDCA similar to that of native GABA(A receptors. The mutation α1V256S, known to reduce the inhibitory action of pregnenolone sulphate, reduced the potency of UDCA. The mutation α1Q241L, which abolishes GABA(AR potentiation by several neurosteroids, had no effect on GABA(AR inhibition by UDCA. In conclusion, UDCA enhances alertness through disinhibition, at least partially of the histaminergic system via GABA(A receptors.

  11. Dynamic mobility of functional GABAA receptors at inhibitory synapses.

    Science.gov (United States)

    Thomas, Philip; Mortensen, Martin; Hosie, Alastair M; Smart, Trevor G

    2005-07-01

    Importing functional GABAA receptors into synapses is fundamental for establishing and maintaining inhibitory transmission and for controlling neuronal excitability. By introducing a binding site for an irreversible inhibitor into the GABAA receptor alpha1 subunit channel lining region that can be accessed only when the receptor is activated, we have determined the dynamics of receptor mobility between synaptic and extrasynaptic locations in hippocampal pyramidal neurons. We demonstrate that the cell surface GABAA receptor population shows no fast recovery after irreversible inhibition. In contrast, after selective inhibition, the synaptic receptor population rapidly recovers by the import of new functional entities within minutes. The trafficking pathways that promote rapid importation of synaptic receptors do not involve insertion from intracellular pools, but reflect receptor diffusion within the plane of the membrane. This process offers the synapse a rapid mechanism to replenish functional GABAA receptors at inhibitory synapses and a means to control synaptic efficacy.

  12. Chronic prenatal ethanol exposure alters hippocampal GABA(A) receptors and impairs spatial learning in the guinea pig.

    Science.gov (United States)

    Iqbal, U; Dringenberg, H C; Brien, J F; Reynolds, J N

    2004-04-02

    Chronic prenatal ethanol exposure (CPEE) can injure the developing brain, and may lead to the fetal alcohol syndrome (FAS). Previous studies have demonstrated that CPEE upregulates gamma-aminobutyric acid type A (GABA(A)) receptor expression in the cerebral cortex, and decreases functional synaptic plasticity in the hippocampus, in the adult guinea pig. This study tested the hypothesis that CPEE increases GABA(A) receptor expression in the hippocampus of guinea pig offspring that exhibit cognitive deficits in a hippocampal-dependent spatial learning task. Timed, pregnant guinea pigs were treated with ethanol (4 g/kg maternal body weight per day), isocaloric-sucrose/pair-feeding, or water throughout gestation. GABA(A) receptor subunit protein expression in the hippocampus was measured at two development ages: near-term fetus and young adult. In young adult guinea pig offspring, CPEE increased spontaneous locomotor activity in the open-field and impaired task acquisition in the Morris water maze. CPEE did not change GABA(A) receptor subunit protein expression in the near-term fetal hippocampus, but increased expression of the beta2/3-subunit of the GABA(A) receptor in the hippocampus of young adult offspring. CPEE did not change either [(3)H]flunitrazepam binding or GABA potentiation of [(3)H]flunitrazepam binding, but decreased the efficacy of allopregnanolone potentiation of [(3)H]flunitrazepam binding, to hippocampal GABA(A) receptors in adult offspring. Correlational analysis revealed a relationship between increased spontaneous locomotor activity and growth restriction in the hippocampus induced by CPEE. Similarly, an inverse relationship was found between performance in the water maze and the efficacy of allopregnanolone potentiation of [(3)H]flunitrazepam binding in the hippocampus. These data suggest that alterations in hippocampal GABA(A) receptor expression and pharmacological properties contribute to hippocampal-related behavioral and cognitive deficits

  13. Aripiprazole Increases the PKA Signalling and Expression of the GABAA Receptor and CREB1 in the Nucleus Accumbens of Rats.

    Science.gov (United States)

    Pan, Bo; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2016-05-01

    The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling.

  14. Positron Emission Tomography (PET Quantification of GABAA Receptors in the Brain of Fragile X Patients.

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    Charlotte D'Hulst

    Full Text Available Over the last several years, evidence has accumulated that the GABAA receptor is compromised in animal models for fragile X syndrome (FXS, a common hereditary form of intellectual disability. In mouse and fly models, agonists of the GABAA receptor were able to rescue specific consequences of the fragile X mutation. Here, we imaged and quantified GABAA receptors in vivo in brain of fragile X patients using Positron Emission Topography (PET and [11C]flumazenil, a known high-affinity and specific ligand for the benzodiazepine site of GABAA receptors. We measured regional GABAA receptor availability in 10 fragile X patients and 10 control subjects. We found a significant reduction of on average 10% in GABAA receptor binding potential throughout the brain in fragile X patients. In the thalamus, the brain region showing the largest difference, the GABAA receptor availability was even reduced with 17%. This is one of the first reports of a PET study of human fragile X brain and directly demonstrates that the GABAA receptor availability is reduced in fragile X patients. The study reinforces previous hypotheses that the GABAA receptor is a potential target for rational pharmacological treatment of fragile X syndrome.

  15. Context-Dependent Modulation of αβγ and αβγ GABAA Receptors by Penicillin: Implications for Phasic and Tonic Inhibition

    Science.gov (United States)

    Feng, Hua-Jun; Botzolakis, Emmanuel J.; Macdonald, Robert L.

    2009-01-01

    Summary Penicillin, an open-channel blocker of GABAA receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABAA receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents evoked from both synaptic and extrasynaptic receptor isoforms. However, penicillin had isoform-specific effects on the extent of desensitization, reflecting its ability to differentially modulate peak (non-equilibrium) and residual (near-equilibrium) currents. This suggested that the context of activation could determine the apparent sensitivity of a given receptor isoform to penicillin. To test this hypothesis, we explored the ability of penicillin to modulate synaptic and extrasynaptic isoforms that were activated under more physiologically relevant conditions. Interestingly, while currents evoked from synaptic isoforms under phasic conditions (transient activation by a saturating concentration of GABA) were substantially inhibited by penicillin, currents evoked from extrasynaptic isoforms under tonic conditions (prolonged application by a sub-saturating concentration of GABA) were minimally affected. We therefore concluded that the reported inability of penicillin to modulate tonic currents could not simply be attributed to insensitivity of extrasynaptic receptors, but rather, reflected an inability to modulate these receptors in their native context of activation. PMID:18775733

  16. γ1-Containing GABA-A Receptors Cluster at Synapses Where they Mediate Slower Synaptic Currents than γ2-Containing GABA-A Receptors

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    Christine L. Dixon

    2017-06-01

    Full Text Available GABA-A receptors (GABAARs are pentameric ligand-gated ion channels that are assembled mainly from α (α1–6, β (β1–3 and γ (γ1–3 subunits. Although GABAARs containing γ2L subunits mediate most of the inhibitory neurotransmission in the brain, significant expression of γ1 subunits is seen in the amygdala, pallidum and substantia nigra. However, the location and function of γ1-containing GABAARs in these regions remains unclear. In “artificial” synapses, where the subunit composition of postsynaptic receptors is specifically controlled, γ1 incorporation slows the synaptic current decay rate without affecting channel deactivation, suggesting that γ1-containing receptors are not clustered and therefore activated by diffuse neurotransmitter. However, we show that γ1-containing receptors are localized at neuronal synapses and form clusters in both synaptic and extrasynaptic regions. In addition, they exhibit rapid membrane diffusion and a higher frequency of exchange between synaptic and perisynaptic populations compared to γ2L-containing GABAARs. A point mutation in the large intracellular domain and a pharmacological analysis reveal that when a single non-conserved γ2L residue is mutated to its γ1 counterpart (T349L, the synaptic current decay is slowed from γ2L- to γ1-like without changing the clustering or diffusion properties of the receptors. In addition, previous fast perfusion and single channel kinetic experiments revealed no difference in the intrinsic closing rates of γ2L- and γ1-containing receptors when expressed in HEK293 cells. These observations together with Monte Carlo simulations of synaptic function confirm that decreased clustering does not control γ1-containing GABAAR kinetics. Rather, they suggest that γ1- and γ2L-containing receptors exhibit differential synaptic current decay rates due to differential gating dynamics when localized at the synapse.

  17. GABAA Receptors, Anesthetics and Anticonvulsants in Brain Development

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    Henschel, Oliver; Gipson, Keith E.; Bordey, Angelique

    2008-01-01

    GABA, acting via GABAA receptors, is well-accepted as the main inhibitory neurotransmitter of the mature brain, where it dampens neuronal excitability. The receptor's properties have been studied extensively, yielding important information about its structure, pharmacology, and regulation that are summarized in this review. Several GABAergic drugs have been commonly used as anesthetics, sedatives, and anticonvulsants for decades. However, findings that GABA has critical functions in brain development, in particular during the late embryonic and neonatal period, raise worthwhile questions regarding the side effects of GABAergic drugs that may lead to long-term cognitive deficits. Here, we will review some of these drugs in parallel with the control of CNS development that GABA exerts via activation of GABAA receptors. This review aims to provide a basic science and clinical perspective on the function of GABA and related pharmaceuticals acting at GABAA receptors. PMID:18537647

  18. Glutamate AMPA/kainate receptors, not GABA(A) receptors, mediate estradiol-induced sex differences in the hypothalamus.

    Science.gov (United States)

    Todd, Brigitte J; Schwarz, Jaclyn M; Mong, Jessica A; McCarthy, Margaret M

    2007-02-15

    Sex differences in brain morphology underlie physiological and behavioral differences between males and females. During the critical perinatal period for sexual differentiation in the rat, gonadal steroids act in a regionally specific manner to alter neuronal morphology. Using Golgi-Cox impregnation, we examined several parameters of neuronal morphology in postnatal day 2 (PN2) rats. We found that in the ventromedial nucleus of the hypothalamus (VMN) and in areas just dorsal and just lateral to the VMN that there was a sex difference in total dendritic spine number (males greater) that was abolished by treating female neonates with exogenous testosterone. Dendritic branching was similarly sexually differentiated and hormonally modulated in the VMN and dorsal to the VMN. We then used spinophilin, a protein that positively correlates with the amount of dendritic spines, to investigate the mechanisms underlying these sex differences. Estradiol, which mediates most aspects of masculinization and is the aromatized product of testosterone, increased spinophilin levels in female PN2 rats to that of males. Muscimol, an agonist at GABA(A) receptors, did not affect spinophilin protein levels in either male or female neonates. Kainic acid, an agonist at glutamatergic AMPA/kainate receptors, mimicked the effect of estradiol in females. Antagonizing AMPA/kainate receptors with NBQX prevented the estradiol-induced increase in spinophilin in females but did not affect spinophilin level in males. (c) 2007 Wiley Periodicals, Inc.

  19. The Role of GABAA Receptor in Antispasmodic Activity of Hydroalcholic Extract of Petroselinum Crispum (Parsley Seed in Rat Ileum

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    Feryal Savary

    2017-01-01

    Full Text Available Abstract Background: Parsley is one of the medicinal herbs used for gastrointestinal disorders. However, spasmolytic activity of Petroselinum crispum (parsley extract has been reported, there is a lack of information to support the mechanism of this antispasmodic activity. Taking this into account, the purpose of the present work was to investigate the role of GABAA receptor on antispasmodic activity of the hydroalcoholic extract of parsley seed in isolated rat ileum. Materials and Methods: In this study, terminal portion of ileum (2 cm was dissected out and mounted in an organ bath containing air bubbled Tyrode solution (37οC, pH=7.4. Under 1gr resting tension, ileal contraction was induced by KCl (60 mM and recorded isotonically. The effects of non-cumulative (0.1-0.5 mg/ml concentrations of extract on KCl-induced contractions were examined. After evaluating the effect of agonist and antagonist GABAA receptor, the effect of parsley extract was assessed in the presence of muscimol (25 µM and bicuculline (10 µM as agonist and antagonist of GABAA, respectively. Results: Parsley seed extract reduced the KCl-induced ileal contraction in a concentration-dependent manner (n=7, p<0.001. Both muscimol and bicuculline exerted relaxant effect on ileal contraction (n=7, p<0.05, p<0.01, respectively. Surprisingly, agonist and antagonist of GABAA both potentiated the spasmolytic effect of extract (0.2 mg/ml. Altogether, spasmolytic effect of extract was not attenuated in the presence of GABAA antagonist. Conclusion: It seems that GABAA receptor is not involved in the antispasmodic effect of parsley seeds extract in rat ileum.

  20. Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

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    Paul David Whissell

    2013-09-01

    Full Text Available Extrasynaptic γ-aminobutyric acid type A (GABAA receptors that contain the δ subunit (δGABAA receptors are expressed in several brain regions including the dentate gyrus (DG and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p. on memory performance in wild-type (WT and δGABAA receptor null mutant (Gabrd–/– mice. Additionally, the effects of THIP on long-term potentiation (LTP, a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd–/– mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd–/– mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity.

  1. Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

    Science.gov (United States)

    Whissell, Paul D.; Eng, Dave; Lecker, Irene; Martin, Loren J.; Wang, Dian-Shi; Orser, Beverley A.

    2013-01-01

    Extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that contain the δ subunit (δGABAA receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABAA receptor null mutant (Gabrd−/−) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd−/− mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd−/− mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity. PMID:24062648

  2. Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21WAF1/CIP1

    International Nuclear Information System (INIS)

    Kim, Mi Jin; Yoo, Young A.; Kim, Hyung Jung; Kang, Seongman; Kim, Yong Geon; Kim, Jun Suk; Yoo, Young Do

    2005-01-01

    Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27 Kip1 in the absence of p53. This study found that MRPL41 mediates the p21 WAF1/CIP1 -mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21 WAF1/CIP1 and p27 Kip1 levels under the growth inhibitory conditions

  3. Identification of a novel protein complex containing ASIC1a and GABAA receptors and their interregulation.

    Directory of Open Access Journals (Sweden)

    Dongbo Zhao

    Full Text Available Acid-sensing ion channels (ASICs belong to the family of the epithelial sodium channel/degenerin (ENaC/DEG and are activated by extracellular protons. They are widely distributed within both the central and peripheral nervous systems. ASICs were modified by the activation of γ-aminobutyric acid receptors (GABAA, a ligand-gated chloride channels, in hippocampal neurons. In contrast, the activity of GABAA receptors were also modulated by extracellular pH. However so far, the mechanisms underlying this intermodulation remain obscure. We hypothesized that these two receptors-GABAA receptors and ASICs channels might form a novel protein complex and functionally interact with each other. In the study reported here, we found that ASICs were modified by the activation of GABAA receptors either in HEK293 cells following transient co-transfection of GABAA and ASIC1a or in primary cultured dorsal root ganglia (DRG neurons. Conversely, activation of ASIC1a also modifies the GABAA receptor-channel kinetics. Immunoassays showed that both GABAA and ASIC1a proteins were co-immunoprecipitated mutually either in HEK293 cells co-transfected with GABAA and ASIC1a or in primary cultured DRG neurons. Our results indicate that putative GABAA and ASIC1a channels functionally interact with each other, possibly via an inter-molecular association by forming a novel protein complex.

  4. The tuberal lateral hypothalamus is a major target for GABAA--but not GABAB-mediated control of food intake.

    Science.gov (United States)

    Turenius, Christine I; Charles, Jonathan R; Tsai, Donna H; Ebersole, Priscilla L; Htut, Myat H; Ngo, Phuong T; Lara, Raul N; Stanley, B Glenn

    2009-08-04

    The lateral hypothalamus (LH) is a site of integration for control mechanisms of feeding behavior as it has extensive reciprocal connections with multiple intrahypothalamic and extrahypothalamic brain areas. Evidence suggests that blockade of ionotropric gamma-aminobutyric acid (GABA) receptors in the LH elicits eating in satiated rats. To determine whether this GABA(A) receptor antagonist effect is specific to the LH, the antagonist picrotoxin was injected into one of six nearby sites and food intake was measured. Picrotoxin at 133 pmol elicited eating in the LH, but not in surrounding sites (thalamus, lateral preoptic area, ventral tegmental area, dorsomedial hypothalamus, and entopeduncular nucleus). More specifically, picrotoxin injected into the tuberal LH (tLH) elicited eating, but was ineffective when injected into the anterior or posterior LH. We also investigated whether GABA(B) receptors in the LH participated in the control of food intake and found that neither blockade nor activation of these receptors under multiple conditions changed food intake. Collectively, our findings suggest that GABA(A) but not GABA(B) receptors in the tLH act to suppress feeding behavior.

  5. GABA(A) Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

    DEFF Research Database (Denmark)

    Young, Stephanie Z; Platel, Jean-Claude; Nielsen, Jakob V

    2010-01-01

    In the adult neurogenic subventricular zone (SVZ), the behavior of astrocyte-like cells and some of their functions depend on changes in intracellular Ca(2+) levels and tonic GABA(A) receptor activation. However, it is unknown whether, and if so how, GABA(A) receptor activity regulates...... intracellular Ca(2+) dynamics in SVZ astrocytes. To monitor Ca(2+) activity selectively in astrocyte-like cells, we used two lines of transgenic mice expressing either GFP fused to a Gq-coupled receptor or DsRed under the human glial fibrillary acidic protein (hGFAP) promoter. GABA(A) receptor activation...... induced Ca(2+) increases in 40-50% of SVZ astrocytes. GABA(A)-induced Ca(2+) increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC). The L-type Ca(2+) channel activator BayK 8644 increased the percentage of GABA(A)-responding astrocyte...

  6. Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

    Science.gov (United States)

    Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

    1996-11-01

    It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

  7. Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors

    Directory of Open Access Journals (Sweden)

    Milan Fedurco

    2015-01-01

    Full Text Available The California poppy (Eschscholzia californica Cham. contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α1β2γ2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S-reticuline behaved as positive allosteric modulator at the α3, α5, and α6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S-reticuline at the α3β2γ2 and α5β2γ2 GABAA receptors. Interestingly, α1, α3, and α5 were not significantly affected by (R-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine—suspected (S-reticuline metabolites in the rodent brain.

  8. 5-HT1A receptor blockade reverses GABA(A) receptor alpha(3) subunit-mediated anxiolytic effects on stress-induced hyperthermia

    NARCIS (Netherlands)

    Vinkers, Christiaan H.; van Oorschot, Ruud; Korte, S. Mechiel; Olivier, Berend; Groenink, Lucianne

    Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways, and clinically effective anxiolytics act via both neurotransmitter systems. As there is evidence that the GABA(A) and the serotonin receptor system interact, a serotonergic component in the

  9. Region-specificity of GABAA receptor mediated effects on orientation and direction selectivity in cat visual cortical area 18.

    Science.gov (United States)

    Jirmann, Kay-Uwe; Pernberg, Joachim; Eysel, Ulf T

    2009-01-01

    The role of GABAergic inhibition in orientation and direction selectivity has been investigated with the GABA(A)-Blocker bicuculline in the cat visual cortex, and results indicated a region specific difference of functional contributions of GABAergic inhibition in areas 17 and 18. In area 17 inhibition appeared mainly involved in sculpturing orientation and direction tuning, while in area 18 inhibition seemed more closely associated with temporal receptive field properties. However, different types of stimuli were used to test areas 17 and 18 and further studies performed in area 17 suggested an important influence of the stimulus type (single light bars vs. moving gratings) on the evoked responses (transient vs. sustained) and inhibitory mechanisms (GABA(A) vs. GABA(B)) which in turn might be more decisive for the specific results than the cortical region. To insert the missing link in this chain of arguments it was necessary to study GABAergic inhibition in area 18 with moving light bars, which has not been done so far. Therefore, in the present study we investigated area 18 cells responding to oriented moving light bars with extracellular recordings and reversible microiontophoretic blockade of GABAergig inhibition with bicuculline methiodide. The majority of neurons was characterized by a pronounced orientation specificity and variable degrees of direction selectivity. GABA(A)ergic inhibition significantly influenced preferred orientation and preferred direction in area 18. During the action of bicuculline orientation tuning width increased and orientation and direction selectivity indices decreased. Our results obtained in area 18 with moving bar stimuli, although in the proportion of affected cells similar to those described in area 17, quantitatively matched the findings for direction and orientation specificity obtained with moving gratings in area 18. Accordingly, stimulus type is not decisive in area 18 and the GABA(A) dependent, inhibitory intracortical

  10. A novel GABA(A) alpha 5 receptor inhibitor with therapeutic potential.

    Science.gov (United States)

    Ling, István; Mihalik, Balázs; Etherington, Lori-An; Kapus, Gábor; Pálvölgyi, Adrienn; Gigler, Gábor; Kertész, Szabolcs; Gaál, Attila; Pallagi, Katalin; Kiricsi, Péter; Szabó, Éva; Szénási, Gábor; Papp, Lilla; Hársing, László G; Lévay, György; Spedding, Michael; Lambert, Jeremy J; Belelli, Delia; Barkóczy, József; Volk, Balázs; Simig, Gyula; Gacsályi, István; Antoni, Ferenc A

    2015-10-05

    Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. SERUM ACETYLCHOLINESTERASE LEVEL IN THE PATIENTS OF OPIOID (BROWN SUGAR) DEPENDENCE

    OpenAIRE

    Shah, Nilesh; Dave, Kirti

    1992-01-01

    The authors compared the serum acetylcholinesterase level in the patients of brown sugar dependence and the normal volunteers. Significantly lower level of serum acetylcholinesterase was found in patients of brown sugar dependence.

  12. GABAA receptor binding molecules from traditional Chinese Medicine: An in silico approach

    Directory of Open Access Journals (Sweden)

    Meng Lv

    2014-12-01

    Full Text Available The advents of how anesthesia works have helped in the discovery of anesthetic target protein. One such target protein named ϒ-aminobutyric acid (GABAA, which is the chief inhibitory neurotransmitter in the mammalian central nervous system. Asparagine at 289 position of GABAA protein within TM2 is important for its anesthetic function. This study explores Traditional Chinese Medicine (TCM against ASN 289 of GABAA for novel anesthetic compounds. The in silico approach showed gastrodin out of all compounds to be the best compound to start further analysis. It is a potential anesthetic compound suitable for the development of new drug.

  13. Tonically Active α5GABAA Receptors Reduce Motoneuron Excitability and Decrease the Monosynaptic Reflex

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    Martha Canto-Bustos

    2017-09-01

    Full Text Available Motoneurons, the final common path of the Central Nervous System (CNS, are under a complex control of its excitability in order to precisely translate the interneuronal pattern of activity into skeletal muscle contraction and relaxation. To fulfill this relevant function, motoneurons are provided with a vast repertoire of receptors and channels, including the extrasynaptic GABAA receptors which have been poorly investigated. Here, we confirmed that extrasynaptic α5 subunit-containing GABAA receptors localize with choline acetyltransferase (ChAT positive cells, suggesting that these receptors are expressed in turtle motoneurons as previously reported in rodents. In these cells, α5GABAA receptors are activated by ambient GABA, producing a tonic shunt that reduces motoneurons’ membrane resistance and affects their action potential firing properties. In addition, α5GABAA receptors shunted the synaptic excitatory inputs depressing the monosynaptic reflex (MSR induced by activation of primary afferents. Therefore, our results suggest that α5GABAA receptors may play a relevant physiological role in motor control.

  14. Memory Deficits Induced by Inflammation Are Regulated by α5-Subunit-Containing GABAA Receptors

    Directory of Open Access Journals (Sweden)

    Dian-Shi Wang

    2012-09-01

    Full Text Available Systemic inflammation causes learning and memory deficits through mechanisms that remain poorly understood. Here, we studied the pathogenesis of memory loss associated with inflammation and found that we could reverse memory deficits by pharmacologically inhibiting α5-subunit-containing γ-aminobutyric acid type A (α5GABAA receptors and deleting the gene associated with the α5 subunit. Acute inflammation reduces long-term potentiation, a synaptic correlate of memory, in hippocampal slices from wild-type mice, and this reduction was reversed by inhibition of α5GABAA receptor function. A tonic inhibitory current generated by α5GABAA receptors in hippocampal neurons was increased by the key proinflammatory cytokine interleukin-1β through a p38 mitogen-activated protein kinase signaling pathway. Interleukin-1β also increased the surface expression of α5GABAA receptors in the hippocampus. Collectively, these results show that α5GABAA receptor activity increases during inflammation and that this increase is critical for inflammation-induced memory deficits.

  15. Serum inflammatory mediators as markers of human Lyme disease activity.

    Directory of Open Access Journals (Sweden)

    Mark J Soloski

    Full Text Available Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005 in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG, CXCL10 (IP-10 and CCL19 (MIP3B were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively. There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01 and the decrease correlates with chemokine levels (p = 0.0375. The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

  16. Serum Inflammatory Mediators as Markers of Human Lyme Disease Activity

    Science.gov (United States)

    Soloski, Mark J.; Crowder, Lauren A.; Lahey, Lauren J.; Wagner, Catriona A.

    2014-01-01

    Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (pLyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations. PMID:24740099

  17. Activation of Glycine and Extrasynaptic GABAA Receptors by Taurine on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis

    Science.gov (United States)

    Bhattarai, Janardhan Prasad; Park, Soo Joung; Han, Seong Kyu

    2013-01-01

    The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC) with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 μM to 3 mM) showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 μM and 723 μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 μM) and almost completely blocked by strychnine (2 μM), suggesting that taurine-mediated responses are via glycine receptor (GlyR) activation. In addition, taurine (1 mM) activated extrasynaptic GABAA receptor (GABAAR)-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABAARs on the SG neurons. PMID:24379976

  18. Activation of Glycine and Extrasynaptic GABAA Receptors by Taurine on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis

    Directory of Open Access Journals (Sweden)

    Thi Thanh Hoang Nguyen

    2013-01-01

    Full Text Available The substantia gelatinosa (SG of the trigeminal subnucleus caudalis (Vc has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 μM to 3 mM showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 μM and 723 μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 μM and almost completely blocked by strychnine (2 μM, suggesting that taurine-mediated responses are via glycine receptor (GlyR activation. In addition, taurine (1 mM activated extrasynaptic GABAA receptor (GABAAR-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABAARs on the SG neurons.

  19. Anti-thymocyte serum as part of an immunosuppressive regimen in treating haematological immune-mediated diseases in dogs.

    Science.gov (United States)

    Cuq, B; Blois, S L; Mathews, K A

    2017-06-01

    To report the outcomes associated with the use of rabbit anti-dog thymocyte serum in dogs with haematological immune-mediated diseases. Medical records from 2000 to 2016 of patients diagnosed with immune-mediated haemolytic anaemia, immune-mediated thrombocytopenia, pancytopenia and myelofibrosis were reviewed. All dogs had a severe or refractory disease and received rabbit anti-dog thymocyte serum. Lymphocyte counts were used to monitor the immediate anti-thymocyte effect of therapy; long-term patient outcome was recorded. A total of 10 dogs were included. All dogs except one had a notable decrease in their lymphocyte count after rabbit anti-dog thymocyte serum; four of nine had a decrease to less than 10% of the initial lymphocyte count and one dog reached 10·8%. All dogs were discharged from the hospital following their treatment. The dog with no alteration of lymphocyte count following therapy with rabbit anti-dog thymocyte serum had refractory immune mediated haemolytic anemia and was euthanised within two weeks. All other cases achieved clinical remission with immunosuppressive therapy eventually being tapered (3 of 10) or discontinued (6 of 10). Rabbit anti-dog thymocyte serum therapy might be of interest as an adjunctive therapy in refractory immune-mediated diseases and suppressed lymphocyte counts in most dogs. © 2017 British Small Animal Veterinary Association.

  20. In silico comparative genomic analysis of GABAA receptor transcriptional regulation

    Directory of Open Access Journals (Sweden)

    Joyce Christopher J

    2007-06-01

    Full Text Available Abstract Background Subtypes of the GABAA receptor subunit exhibit diverse temporal and spatial expression patterns. In silico comparative analysis was used to predict transcriptional regulatory features in individual mammalian GABAA receptor subunit genes, and to identify potential transcriptional regulatory components involved in the coordinate regulation of the GABAA receptor gene clusters. Results Previously unreported putative promoters were identified for the β2, γ1, γ3, ε, θ and π subunit genes. Putative core elements and proximal transcriptional factors were identified within these predicted promoters, and within the experimentally determined promoters of other subunit genes. Conserved intergenic regions of sequence in the mammalian GABAA receptor gene cluster comprising the α1, β2, γ2 and α6 subunits were identified as potential long range transcriptional regulatory components involved in the coordinate regulation of these genes. A region of predicted DNase I hypersensitive sites within the cluster may contain transcriptional regulatory features coordinating gene expression. A novel model is proposed for the coordinate control of the gene cluster and parallel expression of the α1 and β2 subunits, based upon the selective action of putative Scaffold/Matrix Attachment Regions (S/MARs. Conclusion The putative regulatory features identified by genomic analysis of GABAA receptor genes were substantiated by cross-species comparative analysis and now require experimental verification. The proposed model for the coordinate regulation of genes in the cluster accounts for the head-to-head orientation and parallel expression of the α1 and β2 subunit genes, and for the disruption of transcription caused by insertion of a neomycin gene in the close vicinity of the α6 gene, which is proximal to a putative critical S/MAR.

  1. Enhanced GABAA-Mediated Tonic Inhibition in Auditory Thalamus of Rats with Behavioral Evidence of Tinnitus.

    Science.gov (United States)

    Sametsky, Evgeny A; Turner, Jeremy G; Larsen, Deb; Ling, Lynne; Caspary, Donald M

    2015-06-24

    Accumulating evidence suggests a role for inhibitory neurotransmitter dysfunction in the pathology of tinnitus. Opposing hypotheses proposed either a pathologic decrease or increase of GABAergic inhibition in medial geniculate body (MGB). In thalamus, GABA mediates fast synaptic inhibition via synaptic GABAA receptors (GABAARs) and persistent tonic inhibition via high-affinity extrasynaptic GABAARs. Given that extrasynaptic GABAARs control the firing mode of thalamocortical neurons, we examined tonic GABAAR currents in MGB neurons in vitro, using the following three groups of adult rats: unexposed control (Ctrl); sound exposed with behavioral evidence of tinnitus (Tin); and sound exposed with no behavioral evidence of tinnitus (Non-T). Tonic GABAAR currents were evoked using the selective agonist gaboxadol. Months after a tinnitus-inducing sound exposure, gaboxadol-evoked tonic GABAAR currents showed significant tinnitus-related increases contralateral to the sound exposure. In situ hybridization studies found increased mRNA levels for GABAAR δ-subunits contralateral to the sound exposure. Tin rats showed significant increases in the number of spikes per burst evoked using suprathreshold-injected current steps. In summary, we found little evidence of tinnitus-related decreases in GABAergic neurotransmission. Tinnitus and chronic pain may reflect thalamocortical dysrhythmia, which results from abnormal theta-range resonant interactions between thalamus and cortex, due to neuronal hyperpolarization and the initiation of low-threshold calcium spike bursts (Walton and Llinás, 2010). In agreement with this hypothesis, we found tinnitus-related increases in tonic extrasynaptic GABAAR currents, in action potentials/evoked bursts, and in GABAAR δ-subunit gene expression. These tinnitus-related changes in GABAergic function may be markers for tinnitus pathology in the MGB. Copyright © 2015 the authors 0270-6474/15/359369-12$15.00/0.

  2. Anxiogenic properties of an inverse agonist selective for α3 subunit-containing GABAA receptors

    OpenAIRE

    Atack, John R; Hutson, Peter H; Collinson, Neil; Marshall, George; Bentley, Graham; Moyes, Christopher; Cook, Susan M; Collins, Ian; Wafford, Keith; McKernan, Ruth M; Dawson, Gerard R

    2005-01-01

    α3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype.α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15-1788 in vivo bi...

  3. Differential Modulation of GABAA and NMDA Receptors by an α7-nicotinic Acetylcholine Receptor Agonist in Chronic Glaucoma

    Directory of Open Access Journals (Sweden)

    Xujiao Zhou

    2017-12-01

    Full Text Available Presynaptic modulation of γ-aminobutyric acid (GABA release by an alpha7 nicotinic acetylcholine receptor (α7-nAChR agonist promotes retinal ganglion cell (RGC survival and function, as suggested by a previous study on a chronic glaucomatous model from our laboratory. However, the role of excitatory and inhibitory amino acid receptors and their interaction with α7-nAChR in physiological and glaucomatous events remains unknown. In this study, we investigated GABAA and N-methyl-D-aspartate (NMDA receptor activity in control and glaucomatous retinal slices and the regulation of amino acid receptor expression and function by α7-nAChR. Whole-cell patch-clamp recordings from RGCs revealed that the α7-nAChR specific agonist PNU-282987 enhanced the amplitude of currents elicited by GABA and reduced the amplitude of currents elicited by NMDA. The positive modulation of GABAA receptor and the negative modulation of NMDA receptor (NMDAR by PNU-282987-evoked were prevented by pre-administration of the α7-nAChR antagonist methyllycaconitine (MLA. The frequency and the amplitude of glutamate receptor-mediated miniature glutamatergic excitatory postsynaptic currents (mEPSCs were not significantly different between the control and glaucomatous RGCs. Additionally, PNU-282987-treated slices showed no alteration in the frequency or amplitude of mEPSCs relative to control RGCs. Moreover, we showed that expression of the α1 subunit of the GABAA receptor was downregulated and the expression of the NMDAR NR2B subunit was upregulated by intraocular pressure (IOP elevation, and the changes of high IOP were blocked by PNU-282987. In conclusion, retina GABAA and NMDARs are modulated positively and negatively, respectively, by activation of α7-nAChR in in vivo chronic glaucomatous models.

  4. Ameliorating Role Exerted by Al-Hijamah in Autoimmune Diseases: Effect on Serum Autoantibodies and Inflammatory Mediators

    Science.gov (United States)

    Baghdadi, Hussam; Abdel-Aziz, Nada; Ahmed, Nagwa Sayed; Mahmoud, Hany Salah; Barghash, Ayman; Nasrat, Abdullah; Nabo, Manal Mohamed Helmy; El Sayed, Salah Mohamed

    2015-01-01

    Autoimmune diseases have common properties characterized by abnormal blood chemistry with high serum autoimmune antibodies, and inflammatory mediators. Those causative pathological substances (CPS) cannot be excreted by physiological mechanisms. Current treatments for autoimmune diseases involve steroids, cytotoxic drugs, plasmapheresis and monoclonal antibodies. Wet cupping therapy (WCT) of prophetic medicine is called Al-hijamah that treats numerous diseases having different etiology and pathogenesis via a pressure-dependent and size-dependent non-specific filtration then excretion of CPS causing clearance of blood and interstitial fluids. Al-hijamah clears blood passing through the fenestrated skin capillaries. Medical bases of Al-hijamah were reported in the evidence-based Taibah mechanism (Taibah theory). Al-hijamah was reported to be an excellent treatment for rheumatoid arthritis that improved patients’ blood chemistry and induced significant clinical improvement and pharmacological potentiation. Al-hijamah improved the natural immunity and suppressed the pathological immunity through decreasing the serum level of autoantibodies, inflammatory mediators, and serum ferritin (a key player in autoimmunity). Al-hijamah reduced significantly pain severity, number of swollen joints and disease activity with no significant side effects. Main steps of Al-hijamah are skin suction (cupping), scarification (sharatmihjam in Arabic) and second suction (triple S technique) that is better therapeutically than the traditional WCT (double S technique). Whenever an excess noxious substance is to be removed from patients’ blood and interstitial fluids, Al-hijamah is indicated. Shartatmihjam is a curative treatment in prophetic teachings according to the prophetic hadeeth: “Cure is in three: in shartatmihjam, oral honey and cauterization. I do not recommend my nation to cauterize”. Al-hijamah may have better therapeutic benefits than plasmapheresis. Al-hijamah may be

  5. Washing-free heterogeneous immunosensor using proximity-dependent electron mediation between an enzyme label and an electrode.

    Science.gov (United States)

    Dutta, Gorachand; Kim, Sinyoung; Park, Seonhwa; Yang, Haesik

    2014-05-06

    Washing processes, essential in most heterogeneous labeled assays, have been a big hurdle in simplifying the detection procedure and reducing assay time. Nevertheless, less attention has been paid to washing-free heterogeneous labeled assays. We report a purely washing-free immunosensor that allows fast, sensitive, and single-step detection of prostate-specific antigen in serum with low interference. Proximity-dependent electron mediation of ferrocenemethanol (Fc) between an indium-tin oxide (ITO) electrode and a glucose-oxidase (GOx) label allows us to discriminate between a bound and an unbound label: a bound label offers faster electron mediation than an unbound one. The electrooxidation of Fc at a low applied potential (0.13 V vs Ag/AgCl) and a low electrocatalytic ITO electrode and the oxidation of l-ascorbic acid by l-ascorbate oxidase minimize the effect of the interfering species. With a high concentration of glucose (200 mM), the signal and background levels are hardly dependent on the glucose-concentration variation in the sample. The washing-free immunosensor can detect a concentration of ca. 1 pg/mL for mouse IgG in phosphate-buffered saline and a concentration of ca. 10 pg/mL for prostate-specific antigen spiked in female serum after an incubation period of 10 min. The concentrations measured with actual clinical serum samples are in good agreement with the concentrations measured with a commercial instrument, which renders the washing-free heterogeneous immunosensor appealing for practical use.

  6. Metabolic products of linalool and modulation of GABAA receptors

    Science.gov (United States)

    Milanos, Sinem; Elsharif, Shaimaa A.; Janzen, Dieter; Buettner, Andrea; Villmann, Carmen

    2017-06-01

    Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory a1b2 GABAA receptors in various expression systems. However, in plants or humans, i.e. following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at a1b2g2 GABAA receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC5-10 together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself.

  7. Metabolic Products of Linalool and Modulation of GABAA Receptors

    Directory of Open Access Journals (Sweden)

    Sinem Milanos

    2017-06-01

    Full Text Available Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory α1β2 GABAA receptors in various expression systems. However, in plants or humans, i.e., following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at α1β2γ2 GABAA receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC10−30 together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself.

  8. O2-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    International Nuclear Information System (INIS)

    Ascenzi, Paolo; Gullotta, Francesca; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2011-01-01

    Research highlights: → Human serum heme-albumin displays globin-like properties. → O 2 -mediated oxidation of ferrous nitrosylated human serum heme-albumin. → Allosteric modulation of human serum heme-albumin reactivity. → Rifampicin is an allosteric effector of human serum heme-albumin. → Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O 2 -mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O 2 -mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10 -5 and 8.3 x 10 -4 s -1 , and h = 1.3 x 10 -4 and 8.5 x 10 -4 s -1 , in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 o C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O 2 -mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O 2 does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O 2 -mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  9. Correlation between serum albumin, prealbumin and ventilator dependence in patients with COPD

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    Shu-Miao Wu

    2016-04-01

    Full Text Available Objective: To investigate the correlation between the serum albumin, prealbumin and ventilator dependence in patients with COPD. Methods: Serum albumin and prealbumin of 44 COPD patients using breathing machine>48 h were tested at admission, 1 week, 2 weeks treatment. According to the existence of ventilator dependence, divided the patients into the ventilator dependence group and the ventilator independence group, compare the albumin, prealbumin average of two groups, evaluate the correlation between the serum albumin, prealbumin and ventilator dependence in patients with COPD. Results: Albumin levels reduced in both groups after 1 week treatment, but there was no statistically significant difference. Albumin levels reduced in both groups after 2 weeks treatment, but more significantly in the ventilator dependence group, the difference had statistical significance. Prealbumin levels in the ventilator independence group after 1 week treatment didn’t reduced, but reduced in the ventilator dependence group, the difference was statistically significant. Prealbumin levels reduced in both groups after 2 weeks treatment, but more significant in the ventilator dependence group, the difference had statistical significance. Conclusion: Serum albumin and prealbumin levels were correlation with the ventilator dependence in patients with COPD. But prealbumin can more sensitively and more early predict the ventilator dependence in patients with COPD suffered from malnutrition.

  10. LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

    Science.gov (United States)

    Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

    2012-01-01

    GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

  11. The role of gamma-aminobutyric acid/glycinergic synaptic transmission in mediating bilirubin-induced hyperexcitation in developing auditory neurons.

    Science.gov (United States)

    Yin, Xin-Lu; Liang, Min; Shi, Hai-Bo; Wang, Lu-Yang; Li, Chun-Yan; Yin, Shan-Kai

    2016-01-05

    Hyperbilirubinemia is a common clinical phenomenon observed in human newborns. A high level of bilirubin can result in severe jaundice and bilirubin encephalopathy. However, the cellular mechanisms underlying bilirubin excitotoxicity are unclear. Our previous studies showed the action of gamma-aminobutyric acid (GABA)/glycine switches from excitatory to inhibitory during development in the ventral cochlear nucleus (VCN), one of the most sensitive auditory nuclei to bilirubin toxicity. In the present study, we investigated the roles of GABAA/glycine receptors in the induction of bilirubin hyperexcitation in early developing neurons. Using the patch clamp technique, GABAA/glycine receptor-mediated spontaneous inhibitory synaptic currents (sIPSCs) were recorded from bushy and stellate cells in acute brainstem slices from young mice (postnatal day 2-6). Bilirubin significantly increased the frequency of sIPSCs, and this effect was prevented by pretreatments of slices with either fast or slow Ca(2+) chelators BAPTA-AM and EGTA-AM suggesting that bilirubin can increase the release of GABA/glycine via Ca(2+)-dependent mechanisms. Using cell-attached recording configuration, we found that antagonists of GABAA and glycine receptors strongly attenuated spontaneous spiking firings in P2-6 neurons but produced opposite effect in P15-19 neurons. Furthermore, these antagonists reversed bilirubin-evoked hyperexcitability in P2-6 neurons, indicating that excitatory action of GABA/glycinergic transmission specifically contribute to bilirubin-induced hyperexcitability in the early stage of development. Our results suggest that bilirubin-induced enhancement of presynaptic release GABA/Glycine via Ca(2+)-dependent mechanisms may play a critical role in mediating neuronal hyperexcitation associated with jaundice, implicating potential new strategies for predicting, preventing, and treating bilirubin neurotoxicity. Copyright © 2015. Published by Elsevier Ireland Ltd.

  12. GABA_A receptor function is regulated by lipid bilayer elasticity

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Werge, Thomas; Berthelsen, Camilla

    2006-01-01

    ( s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABAA receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown...... reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABAA receptor function is regulated by lipid bilayer elasticity....... PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer....

  13. Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats.

    Science.gov (United States)

    Blacktop, Jordan M; Vranjkovic, Oliver; Mayer, Matthieu; Van Hoof, Matthew; Baker, David A; Mantsch, John R

    2016-03-01

    Stress-induced reinstatement of cocaine seeking requires corticotropin releasing factor (CRF) actions in the ventral tegmental area (VTA). However the mechanisms through which CRF regulates VTA function to promote cocaine use are not fully understood. Here we examined the role of GABAergic neurotransmission in the VTA mediated by GABA-A or GABA-B receptors in the reinstatement of extinguished cocaine seeking by a stressor, uncontrollable intermittent footshock, or bilateral intra-VTA administration of CRF. Rats underwent repeated daily cocaine self-administration (1.0 mg/kg/ing; 14 × 6 h/day) and extinction and were tested for reinstatement in response to footshock (0.5 mA, 0.5" duration, average every 40 s; range 10-70 s) or intra-VTA CRF delivery (500 ng/side) following intra-VTA pretreatment with the GABA-A antagonist, bicuculline, the GABA-B antagonist, 2-hydroxysaclofen or vehicle. Intra-VTA bicuculline (1, 10 or 20 ng/side) failed to block footshock- or CRF-induced cocaine seeking at either dose tested. By contrast, 2-hydroxysaclofen (0.2 or 2 μg/side) prevented reinstatement by both footshock and intra-VTA CRF at a concentration that failed to attenuate food-reinforced lever pressing (45 mg sucrose-sweetened pellets; FR4 schedule) in a separate group of rats. These data suggest that GABA-B receptor-dependent CRF actions in the VTA mediate stress-induced cocaine seeking and that GABA-B receptor antagonists may have utility for the management of stress-induced relapse in cocaine addicts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Different forms of glycine- and GABAA-receptor mediated inhibitory synaptic transmission in mouse superficial and deep dorsal horn neurons

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    Brichta Alan M

    2009-11-01

    Full Text Available Abstract Background Neurons in superficial (SDH and deep (DDH laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR and GABAA-receptors (GABAARs contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood. Methods and Results Here we compare fast inhibitory synaptic transmission in mouse (P17-37 SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23°C. GlyR-mediated mIPSCs were detected in 74% (25/34 and 94% (25/27 of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18. Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 ± 3.9 vs. 64.7 ± 5.0 pA; n = 25 each, decay time was slower (8.5 ± 0.8 vs. 5.5 ± 0.3 ms, and frequency was lower (0.15 ± 0.03 vs. 0.72 ± 0.13 Hz in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 ± 2.4, n = 14 vs. 25. ± 2.0 pA, n = 18 and frequencies (0.21 ± 0.08 vs. 0.18 ± 0.04 Hz in both regions; however, decay times were slower (23.0 ± 3.2 vs. 18.9 ± 1.8 ms in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 ± 1.6 pS, n = 11 vs. 55.7 ± 1.8, n = 8 and GABAARs (22.7 ± 1.7 pS, n = 10 vs. 22.4 ± 2.0 pS, n = 11 in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA, had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 μM reduced GlyR-mediated mIPSC frequency in SDH

  15. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    Science.gov (United States)

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  16. Computational modeling reveals dendritic origins of GABA(A-mediated excitation in CA1 pyramidal neurons.

    Directory of Open Access Journals (Sweden)

    Naomi Lewin

    Full Text Available GABA is the key inhibitory neurotransmitter in the adult central nervous system, but in some circumstances can lead to a paradoxical excitation that has been causally implicated in diverse pathologies from endocrine stress responses to diseases of excitability including neuropathic pain and temporal lobe epilepsy. We undertook a computational modeling approach to determine plausible ionic mechanisms of GABA(A-dependent excitation in isolated post-synaptic CA1 hippocampal neurons because it may constitute a trigger for pathological synchronous epileptiform discharge. In particular, the interplay intracellular chloride accumulation via the GABA(A receptor and extracellular potassium accumulation via the K/Cl co-transporter KCC2 in promoting GABA(A-mediated excitation is complex. Experimentally it is difficult to determine the ionic mechanisms of depolarizing current since potassium transients are challenging to isolate pharmacologically and much GABA signaling occurs in small, difficult to measure, dendritic compartments. To address this problem and determine plausible ionic mechanisms of GABA(A-mediated excitation, we built a detailed biophysically realistic model of the CA1 pyramidal neuron that includes processes critical for ion homeostasis. Our results suggest that in dendritic compartments, but not in the somatic compartments, chloride buildup is sufficient to cause dramatic depolarization of the GABA(A reversal potential and dominating bicarbonate currents that provide a substantial current source to drive whole-cell depolarization. The model simulations predict that extracellular K(+ transients can augment GABA(A-mediated excitation, but not cause it. Our model also suggests the potential for GABA(A-mediated excitation to promote network synchrony depending on interneuron synapse location - excitatory positive-feedback can occur when interneurons synapse onto distal dendritic compartments, while interneurons projecting to the perisomatic

  17. Chlorpromazine-induced hepatotoxicity during inflammation is mediated by TIRAP-dependent signaling pathway in mice

    International Nuclear Information System (INIS)

    Gandhi, Adarsh; Guo, Tao; Shah, Pranav; Moorthy, Bhagavatula; Ghose, Romi

    2013-01-01

    Inflammation is a major component of idiosyncratic adverse drug reactions (IADRs). To understand the molecular mechanism of inflammation-mediated IADRs, we determined the role of the Toll-like receptor (TLR) signaling pathway in idiosyncratic hepatotoxicity of the anti-psychotic drug, chlorpromazine (CPZ). Activation of TLRs recruits the first adaptor protein, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) to the TIR domain of TLRs leading to the activation of the downstream kinase, c-Jun-N-terminal kinase (JNK). Prolonged activation of JNK leads to cell-death. We hypothesized that activation of TLR2 by lipoteichoic acid (LTA) or TLR4 by lipopolysaccharide (LPS) will augment the hepatotoxicity of CPZ by TIRAP-dependent mechanism involving prolonged activation of JNK. Adult male C57BL/6, TIRAP +/+ and TIRAP −/− mice were pretreated with saline, LPS (2 mg/kg) or LTA (6 mg/kg) for 30 min or 16 h followed by CPZ (5 mg/kg) or saline (vehicle) up to 24 h. We found that treatment of mice with CPZ in presence of LPS or LTA leads to ∼ 3–4 fold increase in serum ALT levels, a marked reduction in hepatic glycogen content, significant induction of serum tumor necrosis factor (TNF) α and prolonged JNK activation, compared to LPS or LTA alone. Similar results were observed in TIRAP +/+ mice, whereas the effects of LPS or LTA on CPZ-induced hepatotoxicity were attenuated in TIRAP −/− mice. For the first time, we show that inflammation-mediated hepatotoxicity of CPZ is dependent on TIRAP, and involves prolonged JNK activation in vivo. Thus, TIRAP-dependent pathways may be targeted to predict and prevent inflammation-mediated IADRs. -- Highlights: ► Inflammation augments the toxicity of an idiosyncratic hepatotoxin chlorpromazine. ► Activation of Toll-like receptors by LPS or LTA induces chlorpromazine toxicity. ► Sustained stress kinase (JNK) activation is associated with chlorpromazine toxicity. ► These studies provide novel mechanistic

  18. Chlorpromazine-induced hepatotoxicity during inflammation is mediated by TIRAP-dependent signaling pathway in mice

    Energy Technology Data Exchange (ETDEWEB)

    Gandhi, Adarsh, E-mail: adarsh.gandhi@nih.gov [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Guo, Tao, E-mail: tguo4@jhu.edu [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Shah, Pranav [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Moorthy, Bhagavatula [Baylor College of Medicine, Department of Pediatrics, 1102 Bates Avenue, Suite 530, Houston, TX 77030 (United States); Ghose, Romi, E-mail: rghose@uh.edu [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States)

    2013-02-01

    Inflammation is a major component of idiosyncratic adverse drug reactions (IADRs). To understand the molecular mechanism of inflammation-mediated IADRs, we determined the role of the Toll-like receptor (TLR) signaling pathway in idiosyncratic hepatotoxicity of the anti-psychotic drug, chlorpromazine (CPZ). Activation of TLRs recruits the first adaptor protein, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) to the TIR domain of TLRs leading to the activation of the downstream kinase, c-Jun-N-terminal kinase (JNK). Prolonged activation of JNK leads to cell-death. We hypothesized that activation of TLR2 by lipoteichoic acid (LTA) or TLR4 by lipopolysaccharide (LPS) will augment the hepatotoxicity of CPZ by TIRAP-dependent mechanism involving prolonged activation of JNK. Adult male C57BL/6, TIRAP{sup +/+} and TIRAP{sup −/−} mice were pretreated with saline, LPS (2 mg/kg) or LTA (6 mg/kg) for 30 min or 16 h followed by CPZ (5 mg/kg) or saline (vehicle) up to 24 h. We found that treatment of mice with CPZ in presence of LPS or LTA leads to ∼ 3–4 fold increase in serum ALT levels, a marked reduction in hepatic glycogen content, significant induction of serum tumor necrosis factor (TNF) α and prolonged JNK activation, compared to LPS or LTA alone. Similar results were observed in TIRAP{sup +/+} mice, whereas the effects of LPS or LTA on CPZ-induced hepatotoxicity were attenuated in TIRAP{sup −/−} mice. For the first time, we show that inflammation-mediated hepatotoxicity of CPZ is dependent on TIRAP, and involves prolonged JNK activation in vivo. Thus, TIRAP-dependent pathways may be targeted to predict and prevent inflammation-mediated IADRs. -- Highlights: ► Inflammation augments the toxicity of an idiosyncratic hepatotoxin chlorpromazine. ► Activation of Toll-like receptors by LPS or LTA induces chlorpromazine toxicity. ► Sustained stress kinase (JNK) activation is associated with chlorpromazine toxicity. ► These studies

  19. Roles of taurine-mediated tonic GABAA receptor activation in the radial migration of neurons in the fetal mouse cerebral cortex

    Directory of Open Access Journals (Sweden)

    Tomonori eFurukawa

    2014-03-01

    Full Text Available γ-Aminobutyric acid (GABA depolarizes embryonic cerebrocortical neurons and continuous activation of the GABAA receptor (GABAAR contributes to their tonic depolarization. Although multiple reports have demonstrated a role of GABAAR activation in neocortical development, including in migration, most of these studies have used pharmacological blockers. Herein, we performed in utero electroporation in GABA synthesis-lacking homozygous GAD67-GFP knock-in mice (GAD67GFP/GFP to label neurons born in the ventricular zone. Three days after electroporation, there were no differences in the distribution of labeled cells between the genotypes. The dose-response properties of cells labeled to detect GABA were equivalent among genotypes. However, continuous blockade of GABAAR with the GABAAR antagonist SR95531 accelerated radial migration. This effect of GABAAR blockade in GAD67GFP/GFP mice suggested a role for alternative endogenous GABAAR agonists. Thus, we tested the role of taurine, which is derived from maternal blood but is abundant in the fetal brain. The taurine-evoked currents in labeled cells were mediated by GABAAR. Taurine uptake was blocked by a taurine transporter inhibitor, 2-(guanidinoethanesulfonic acid (GES, and taurine release was blocked by a volume-sensitive anion channel blocker, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl oxobutyric acid (DCPIB, as examined through high-performance liquid chromatography (HPLC. GES increased the extracellular taurine concentration and induced an inward shift of the holding current, which was reversed by SR95531. In a taurine-deficient mouse model, the GABAAR-mediated tonic currents were greatly reduced, and radial migration was accelerated. As the tonic currents were equivalent among the genotypes of GAD67-GFP knock-in mice, taurine, rather than GABA, might play a major role as an endogenous agonist of embryonic tonic GABAAR conductance, regulating the radial migration of neurons in the

  20. Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

    Directory of Open Access Journals (Sweden)

    Paul C.P. Curtin

    2015-03-01

    Full Text Available Prepulse inhibition (PPI is understood as an inhibitory process that attenuates sensory flow during early stages (20-1000ms of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if prepulse inhibition (PPI is mediated by glycine receptors (GlyRs and/or GABAA receptors (GABAARs in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs recorded in the neurons that initiate startle, the Mauthner-cells (M-cell. We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms and rapidly (< 50ms decaying (feed-forward inhibitory process that disrupts PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI. Additionally we observed increases of the evoked postsynaptic potential (PSP peak amplitude (+87.43 ± 21.53%; N=9 and duration (+204 ± 48.91%, N=9. In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested ISIs (20-500 ms, essentially eliminating PPI at ISIs from 20-100 ms. Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N=5 and PSP duration (+284.95 ± 65.64%, N=5. Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs by 15.07 ± 3.21%, N=7 and 16.23 ± 7.08%, N=5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit.

  1. Piroxicam-mediated modulatory action of 5-hydroxytryptamine serves as a "brake" on neuronal excitability in ischemic stroke

    Directory of Open Access Journals (Sweden)

    Pallab Bhattacharya

    2015-01-01

    Full Text Available Our previous studies indicated an increase in extracellular γ-aminobutyric acid (GABA in rodent′s ischemic brain after Piroxicam administration, leading to alleviation of glutamate mediated excitotoxicity through activation of type A GABA receptor (GABAA. This study was to investigate if GABAA activation by Piroxicam affects extracellular 5-hydroxytryptamine or not. High performance liquid chromatography revealed that there was a significant decrease in extracellular 5-hydroxytryptamine release in ischemic cerebral cortex and striatum in Piroxicam pre-treated rat brains. This suggests a probable role of Piroxicam in reducing extracellular 5-hydroxytryptamine release in ischemic cerebral cortex and striatum possibly due to the GABAA activation by Piroxicam.

  2. Opposing roles for GABAA and GABAC receptors in short-term memory formation in young chicks.

    Science.gov (United States)

    Gibbs, M E; Johnston, G A R

    2005-01-01

    The inhibitory neurotransmitter GABA has both inhibitory and enhancing effects on short-term memory for a bead discrimination task in the young chick. Low doses of GABA (1-3 pmol/hemisphere) injected into the multimodal association area of the chick forebrain, inhibit strongly reinforced memory, whereas higher doses (30-100 pmol/hemisphere) enhance weakly reinforced memory. The effect of both high and low doses of GABA is clearly on short-term memory in terms of both the time of injection and in the time that the memory loss occurs. We argue on the basis of relative sensitivities to GABA and to selective GABA receptor antagonists that low doses of GABA act at GABAC receptors (EC50 approximately 1 microM) and the higher doses of GABA act via GABAA receptors (EC50 approximately 10 microM). The selective GABAA receptor antagonist bicuculline inhibited strongly reinforced memory in a dose and time dependent manner, whereas the selective GABAC receptor antagonists TPMPA and P4MPA enhanced weakly reinforced in a dose and time dependent manner. Confirmation that different levels of GABA affect different receptor subtypes was demonstrated by the shift in the GABA dose-response curves to the selective antagonists. It is clear that GABA is involved in the control of short-term memory formation and its action, enhancing or inhibiting, depends on the level of GABA released at the time of learning.

  3. Participation of GABAA Chloride Channels in the Anxiolytic-Like Effects of a Fatty Acid Mixture

    Directory of Open Access Journals (Sweden)

    Juan Francisco Rodríguez-Landa

    2013-01-01

    Full Text Available Human amniotic fluid and a mixture of eight fatty acids (FAT-M identified in this maternal fluid (C12:0, lauric acid, 0.9 μg%; C14:0, myristic acid, 6.9 μg%; C16:0, palmitic acid, 35.3 μg%; C16:1, palmitoleic acid, 16.4 μg%; C18:0, stearic acid, 8.5 μg%; C18:1cis, oleic acid, 18.4 μg%; C18:1trans, elaidic acid, 3.5 μg%; C18:2, linoleic acid, 10.1 μg% produce anxiolytic-like effects that are comparable to diazepam in Wistar rats, suggesting the involvement of γ-aminobutyric acid-A (GABAA receptors, a possibility not yet explored. Wistar rats were subjected to the defensive burying test, elevated plus maze, and open field test. In different groups, three GABAA receptor antagonists were administered 30 min before FAT-M administration, including the competitive GABA binding antagonist bicuculline (1 mg/kg, GABAA benzodiazepine antagonist flumazenil (5 mg/kg, and noncompetitive GABAA chloride channel antagonist picrotoxin (1 mg/kg. The FAT-M exerted anxiolytic-like effects in the defensive burying test and elevated plus maze, without affecting locomotor activity in the open field test. The GABAA antagonists alone did not produce significant changes in the behavioral tests. Picrotoxin but not bicuculline or flumazenil blocked the anxiolytic-like effect of the FAT-M. Based on the specific blocking action of picrotoxin on the effects of the FAT-M, we conclude that the FAT-M exerted its anxiolytic-like effects through GABAA receptor chloride channels.

  4. The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development

    NARCIS (Netherlands)

    Stojanovic, Tamara; Capo, Ivan; Aronica, Eleonora; Adle-Biassette, Homa; Höger, Harald; Sieghart, Werner; Kovacs, Gabor G.; Milenkovic, Ivan

    2016-01-01

    γ-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABAA receptor. Nineteen different subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-3) can give rise to multiple receptor subtypes

  5. GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

    Science.gov (United States)

    Cheung, Samantha K; Scott, Kristin

    2017-01-01

    Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

  6. Dual Modulators of GABA-A and Alpha 7 Nicotinic Receptors for Treating Autism

    Science.gov (United States)

    2015-10-01

    AWARD NUMBER: W81XWH-13-1-0144 TITLE: Dual Modulators of GABA-A and Alpha 7 Nicotinic Receptors for Treating Autism PRINCIPAL INVESTIGATOR...SUBTITLE 5a. CONTRACT NUMBER Dual Modulators of GABA-A and Alpha 7 Nicotinic Receptors for Treating Autism 5b. GRANT NUMBER W81XWH-13-1-0144 5c...ABSTRACT Autism spectrum disorder (ASD) is a polygenic signaling disorder that may result, in part, from an imbalance in excitatory and inhibitory

  7. Role of the eosinophil in serum-mediated adherence of equine leukocytes to infective larvae of Strongylus vulgaris.

    Science.gov (United States)

    Klei, T R; Chapman, M R; Dennis, V A

    1992-06-01

    The adherence of equine leukocytes to Strongylus vulgaris infective larvae (L3) in the presence of normal and immune sera was examined in vitro. Immune sera promoted adherence of buffy coat cells from ponies with S. vulgaris-induced eosinophilia (eosinophilic ponies) to S. vulgaris L3. However, eosinophils in the buffy coat cells were the predominant adherent cell type. Studies using leukocyte populations enriched for eosinophils, neutrophils, and mononuclear cells from eosinophilic ponies support the observations using buffy coat cells that eosinophils were the main effector cells. Adherent eosinophils from eosinophilic ponies immobilized L3. Neutrophils were less adherent and did not immobilize L3. Mononuclear cells failed to adhere. Normal eosinophils from strongly-naive ponies did not immobilize S. vulgaris L3 in the presence of immune serum, suggesting the in vivo activation of eosinophils in eosinophilic animals. Immune serum promoted less adherence of buffy coat cells to Strongylus edentatus or mixed species of Cyathostominae L3, suggesting that the serum-mediated cellular adherence phenomenon was species-specific. Normal serum promoted less cellular adherence to S. vulgaris L3 than immune serum. The adherence mediated by normal serum was removed by heat inactivation, suggesting that this nonspecific phenomenon was a complement-mediated reaction. Immune globulins promoted reactions similar to that seen using heat-inactivated immune serum, whereas normal globulins did not promote adherence. Immune globulins absorbed with pieces of S. vulgaris adult worms did not promote the adherence of buffy coat cells to S. vulgaris L3, suggesting that adult and L3 stages share antigens important in this phenomenon that resulted in the removal of specific adherence antibody during absorption.

  8. Effects of common anesthetic agents on [(18)F]flumazenil binding to the GABAA receptor

    DEFF Research Database (Denmark)

    Palner, Mikael; Beinat, Corinne; Banister, Sam

    2016-01-01

    in preclinical imaging studies and clinical imaging studies involving patient populations that do not tolerate relatively longer scan times. The objective of this study was to examine the effects of anesthesia on the binding of [(18)F]flumazenil to GABAA receptors in mice. METHODS: Brain and whole blood...... mice. CONCLUSIONS: Anesthesia has pronounced effects on the binding and blood-brain distribution of [(18)F]flumazenil. Consequently, considerable caution must be exercised in the interpretation of preclinical and clinical PET studies of GABAA receptors involving the use of anesthesia.......BACKGROUND: The availability of GABAA receptor binding sites in the brain can be assessed by positron emission tomography (PET) using the radioligand, [(18)F]flumazenil. However, the brain uptake and binding of this PET radioligand are influenced by anesthetic drugs, which are typically needed...

  9. Progesterone Exerts a Neuromodulatory Effect on Turning Behavior of Hemiparkinsonian Male Rats: Expression of 3α-Hydroxysteroid Oxidoreductase and Allopregnanolone as Suggestive of GABAA Receptors Involvement

    Directory of Open Access Journals (Sweden)

    Roberto Yunes

    2015-01-01

    Full Text Available There is a growing amount of evidence for a neuroprotective role of progesterone and its neuroactive metabolite, allopregnanolone, in animal models of neurodegenerative diseases. By using a model of hemiparkinsonism in male rats, injection of the neurotoxic 6-OHDA in left striatum, we studied progesterone’s effects on rotational behavior induced by amphetamine or apomorphine. Also, in order to find potential explanatory mechanisms, we studied expression and activity of nigrostriatal 3α-hydroxysteroid oxidoreductase, the enzyme that catalyzes progesterone to its active metabolite allopregnanolone. Coherently, we tested allopregnanolone for a possible neuromodulatory effect on rotational behavior. Also, since allopregnanolone is known as a GABAA modulator, we finally examined the action of GABAA antagonist bicuculline. We found that progesterone, in addition to an apparent neuroprotective effect, also increased ipsilateral expression and activity of 3α-hydroxysteroid oxidoreductase. It was interesting to note that ipsilateral administration of allopregnanolone reversed a clear sign of motor neurodegeneration, that is, contralateral rotational behavior. A possible GABAA involvement modulated by allopregnanolone was shown by the blocking effect of bicuculline. Our results suggest that early administration of progesterone possibly activates genomic mechanisms that promote neuroprotection subchronically. This, in turn, could be partially mediated by fast, nongenomic, actions of allopregnanolone acting as an acute modulator of GABAergic transmission.

  10. Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate.

    Science.gov (United States)

    Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K; Liu, Peter; Pantua, Homer; Abbas, Alexander R; Nickerson, Nicholas N; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min; Whitfield, Chris; Kapadia, Sharookh B

    2017-05-23

    Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro , the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for "group 2" capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. IMPORTANCE Uropathogenic E. coli (UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistance in vitro and for complement-mediated bacterial clearance in vivo This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of "group 2" capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for

  11. Hyperalgesic effect induced by barbiturates, midazolam and ethanol: pharmacological evidence for GABA-A receptor involvement

    Directory of Open Access Journals (Sweden)

    M.A.K.F. Tatsuo

    1997-02-01

    Full Text Available The involvement of GABA-A receptors in the control of nociception was studied using the tail-flick test in rats. Non-hypnotic doses of the barbiturates phenobarbital (5-50 mg/kg, pentobarbital (17-33 mg/kg, and thiopental (7.5-30 mg/kg, of the benzodiazepine midazolam (10 mg/kg or of ethanol (0.4-1.6 g/kg administered by the systemic route reduced the latency for the tail-flick response, thus inducing a 'hyperalgesic' state in the animals. In contrast, non-convulsant doses of the GABA-A antagonist picrotoxin (0.12-1.0 mg/kg administered systemically induced an increase in the latency for the tail-flick response, therefore characterizing an 'antinociceptive' state. Previous picrotoxin (0.12 mg/kg treatment abolished the hyperalgesic state induced by effective doses of the barbiturates, midazolam or ethanol. Since phenobarbital, midazolam and ethanol reproduced the described hyperalgesic effect of GABA-A-specific agonists (muscimol, THIP, which is specifically antagonized by the GABA-A antagonist picrotoxin, our results suggest that GABA-A receptors are tonically involved in the modulation of nociception in the rat central nervous system

  12. GABAA receptors in visual and auditory cortex and neural activity changes during basic visual stimulation

    Directory of Open Access Journals (Sweden)

    Pengmin eQin

    2012-12-01

    Full Text Available Recent imaging studies have demonstrated that levels of resting GABA in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABAA receptors, in the changes in brain activity between the eyes closed (EC and eyes open (EO state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: An EO and EC block design, allowing the modelling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [18F]Flumazenil PET measure GABAA receptor binding potentials. It was demonstrated that the local-to-global ratio of GABAA receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABAA receptor binding potential in the visual cortex also predicts the change of functional connectivity between visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABAA receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

  13. Triton X-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABA(A) receptors in Xenopus oocytes

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Ebert, Bjarke; Klaerke, Dan

    2009-01-01

    Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na(+) and L-type Ca(2+) channels and GABA(A) receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its...... by flunitrazepam at alpha(1)beta(3)gamma(2S) receptors. All effects were independent of the presence of a gamma(2S) subunit in the GABA(A) receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA(A) receptor through changes in lipid bilayer elasticity....

  14. Species dependence of [64Cu]Cu-Bis(thiosemicarbazone) radiopharmaceutical binding to serum albumins

    International Nuclear Information System (INIS)

    Basken, Nathan E.; Mathias, Carla J.; Lipka, Alexander E.; Green, Mark A.

    2008-01-01

    Introduction: Interactions of three copper(II) bis(thiosemicarbazone) positron emission tomography radiopharmaceuticals with human serum albumin, and the serum albumins of four additional mammalian species, were evaluated. Methods: 64 Cu-labeled diacetyl bis(N 4 -methylthiosemicarbazonato)copper(II) (Cu-ATSM), pyruvaldehyde bis(N 4 -methylthiosemicarbazonato)copper(II) (Cu-PTSM) and ethylglyoxal bis(thiosemicarbazonato)copper(II) (Cu-ETS) were synthesized and their binding to human, canine, rat, baboon and porcine serum albumins quantified by ultrafiltration. Protein binding was also measured for each tracer in human, porcine, rat and mouse serum. Results: The interaction of these neutral, lipophilic copper chelates with serum albumin is highly compound- and species-dependent. Cu-PTSM and Cu-ATSM exhibit particularly high affinity for human serum albumin (HSA), while the albumin binding of Cu-ETS is relatively insensitive to species. At HSA concentrations of 40 mg/ml, '% free' (non-albumin-bound) levels of radiopharmaceutical were 4.0±0.1%, 5.3±0.2% and 38.6±0.8% for Cu-PTSM, Cu-ATSM and Cu-ETS, respectively. Conclusions: Species-dependent variations in radiopharmaceutical binding to serum albumin may need to be considered when using animal models to predict the distribution and kinetics of these compounds in humans

  15. Effects of central histamine receptors blockade on GABA(A) agonist-induced food intake in broiler cockerels.

    Science.gov (United States)

    Morteza, Zendehdel; Vahhab, Babapour; Hossein, Jonaidi

    2008-02-01

    In this study, the effect of intracerebroventricular (i.c.v) injection of H1, H2 and H3 antagonists on feed intake induced by GABA(A) agonist was evaluated. In Experiment 1, the animals received chloropheniramine, a H1 antagonist and then muscimol, a GABA(A) agonist. In Experiment 2, chickens received famotidine, a H2 receptor antagonist, prior to injection of muscimol. Finally in Experiment 3, the birds were injected with thioperamide, a H3 receptor antagonist and muscimol. Cumulative food intake was measured 15, 30, 45, 60, 90, 120, 150 and 180 min after injections. The results of this study indicated that effects of muscimol on food intake inhibited by pretreatment with chloropheneramine maleate (p GABA(A) receptor interaction on food intake in broiler cockerels.

  16. Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

    Science.gov (United States)

    Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

    1997-06-25

    Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

  17. Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Villumsen, Inge S; Wellendorph, Petrine; Smart, Trevor G

    2015-01-01

    BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasyn......BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence...

  18. Potentiating action of propofol at GABAA receptors of retinal bipolar cells

    DEFF Research Database (Denmark)

    Yue, Lan; Xie, An; Bruzik, Karol S

    2011-01-01

    Purpose. Propofol (2,6-diisopropyl phenol), a widely used systemic anesthetic, is known to potentiate GABA(A) receptor activity in a number of CNS neurons and to produce changes in electroretinographically recorded responses of the retina. However, little is known about propofol's effects...... on specific retinal neurons. The authors investigated the action of propofol on GABA-elicited membrane current responses of retinal bipolar cells, which have both GABA(A) and GABA(C) receptors. Methods. Single, enzymatically dissociated bipolar cells obtained from rat retina were treated with propofol...... + propofol) led to a progressive increase in peak response amplitude and, at higher propofol concentrations, additional changes that included a prolonged time course of response recovery. Pre-exposure of the cell to perfusing propofol typically enhanced the rate of development of potentiation produced...

  19. Pharmacologically Counteracting a Phenotypic Difference in Cerebellar GABAA Receptor Response to Alcohol Prevents Excessive Alcohol Consumption in a High Alcohol-Consuming Rodent Genotype.

    Science.gov (United States)

    Kaplan, Josh Steven; Nipper, Michelle A; Richardson, Ben D; Jensen, Jeremiah; Helms, Melinda; Finn, Deborah Ann; Rossi, David James

    2016-08-31

    Cerebellar granule cell GABAA receptor responses to alcohol vary as a function of alcohol consumption phenotype, representing a potential neural mechanism for genetic predilection for alcohol abuse (Kaplan et al., 2013; Mohr et al., 2013). However, there are numerous molecular targets of alcohol in the cerebellum, and it is not known how they interact to affect cerebellar processing during consumption of socially relevant amounts of alcohol. Importantly, direct evidence for a causative role of the cerebellum in alcohol consumption phenotype is lacking. Here we determined that concentrations of alcohol that would be achieved in the blood after consumption of 1-2 standard units (9 mm) suppresses transmission through the cerebellar cortex in low, but not high, alcohol consuming rodent genotypes (DBA/2J and C57BL/6J mice, respectively). This genotype-selective suppression is mediated exclusively by enhancement of granule cell GABAA receptor currents, which only occurs in DBA/2J mice. Simulating the DBA/2J cellular phenotype in C57BL/6J mice by infusing the GABAA receptor agonist, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol hydrochloride, into cerebellar lobules IV-VI, in vivo, significantly reduced their alcohol consumption and blood alcohol concentrations achieved. 4,5,6,7-Tetrahydroisoxazolo-[5,4-c]pyridine-3-ol hydrochloride infusions also significantly decreased sucrose consumption, but they did not affect consumption of water or general locomotion. Thus, genetic differences in cerebellar response to alcohol contributes to alcohol consumption phenotype, and targeting the cerebellar GABAA receptor system may be a clinically viable therapeutic strategy for reducing excessive alcohol consumption. Alcohol abuse is a leading cause of preventable death and illness; and although alcohol use disorders are 50%-60% genetically determined, the cellular and molecular mechanisms of such genetic influences are largely unknown. Here we demonstrate that genetic differences in

  20. Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.

    Science.gov (United States)

    Zhang, Jianliang; Xue, Fenqin; Chang, Yongchang

    2008-10-01

    GABA receptor (GABAR) types C (GABACR) and A (GABAAR) are both GABA-gated chloride channels that are distinguished by their distinct competitive antagonist properties. The structural mechanism underlying these distinct properties is not well understood. In this study, using previously identified binding residues as a guide, we made individual or combined mutations of nine binding residues in the rho1 GABACR subunit to their counterparts in the alpha1beta2gamma2 GABAAR or reverse mutations in alpha1 or beta2 subunits. The mutants were expressed in Xenopus laevis oocytes and tested for sensitivities of GABA-induced currents to the GABAA and GABAC receptor antagonists. The results revealed that bicuculline insensitivity of the rho1 GABACR was mainly determined by Tyr106, Phe138 and Phe240 residues. Gabazine insensitivity of the rho1 GABACR was highly dependent on Tyr102, Tyr106, and Phe138. The sensitivity of the rho1 GABACR to 3-aminopropyl-phosphonic acid and its analog 3-aminopropyl-(methyl)phosphinic acid mainly depended on residues Tyr102, Val140, FYS240-242, and Phe138. Thus, the residues Tyr102, Tyr106, Phe138, and Phe240 in the rho1 GABACR are major determinants for its antagonist properties distinct from those in the GABAAR. In addition, Val140 in the GABACR contributes to 3-APA binding. In conclusion, we have identified the key structural elements underlying distinct antagonist properties for the GABACR. The mechanistic insights were further extended and discussed in the context of antagonists docking to the homology models of GABAA or GABAC receptors.

  1. GABA(A) receptors in visual and auditory cortex and neural activity changes during basic visual stimulation.

    Science.gov (United States)

    Qin, Pengmin; Duncan, Niall W; Wiebking, Christine; Gravel, Paul; Lyttelton, Oliver; Hayes, Dave J; Verhaeghe, Jeroen; Kostikov, Alexey; Schirrmacher, Ralf; Reader, Andrew J; Northoff, Georg

    2012-01-01

    Recent imaging studies have demonstrated that levels of resting γ-aminobutyric acid (GABA) in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABA(A) receptors, in the changes in brain activity between the eyes closed (EC) and eyes open (EO) state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: an EO and EC block design, allowing the modeling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [(18)F]Flumazenil PET to measure GABA(A) receptor binding potentials. It was demonstrated that the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex also predicted the change in functional connectivity between the visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABA(A) receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

  2. Identification of amino acids involved in histamine potentiation of GABA(A receptors

    Directory of Open Access Journals (Sweden)

    Ulrike eThiel

    2015-05-01

    Full Text Available Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans and rodents, the histaminergic neurons found in the tuberomamillary nucleus (TMN project widely throughout the central nervous system (CNS. Histamine acts as positive modulator of GABA(A receptors (GABA(ARs and, in high concentrations (10 mM, as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABA(ARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABA(ARs. We expressed GABA(ARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues ß2(N265 and ß2(M286, which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues alpha1(R120, ß2(Y157, ß3(D163, ß3(V175 and ß3(Q185. We showed that the amino acid residues ß2(Y157 and ß3(Q185 mediate the positive modulatory effect of histamine on GABA-induced currents, whereas alpha1(R120 and ß2(D163 form a potential histamine interaction site in GABA(ARs.

  3. A Potential Epigenetic Marker Mediating Serum Folate and Vitamin B12 Levels Contributes to the Risk of Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Loo Keat Wei

    2015-01-01

    Full Text Available Stroke is a multifactorial disease that may be associated with aberrant DNA methylation profiles. We investigated epigenetic dysregulation for the methylenetetrahydrofolate reductase (MTHFR gene among ischemic stroke patients. Cases and controls were recruited after obtaining signed written informed consents following a screening process against the inclusion/exclusion criteria. Serum vitamin profiles (folate, vitamin B12, and homocysteine were determined using immunoassays. Methylation profiles for CpGs A and B in the MTHFR gene were determined using a bisulfite-pyrosequencing method. Methylation of MTHFR significantly increased the susceptibility risk for ischemic stroke. In particular, CpG A outperformed CpG B in mediating serum folate and vitamin B12 levels to increase ischemic stroke susceptibility risks by 4.73-fold. However, both CpGs A and B were not associated with serum homocysteine levels or ischemic stroke severity. CpG A is a potential epigenetic marker in mediating serum folate and vitamin B12 to contribute to ischemic stroke.

  4. Time-Dependent Decline in Serum Phenytoin Concentration with Heightened Convulsive Seizure Risk by Prolonged Administration of Fosphenytoin in Japanese: A Retrospective Study.

    Science.gov (United States)

    Ohno, Yuta; Niwa, Takashi; Hirai, Keita; Suzuki, Keiko; Yamada, Yuto; Hayashi, Yuichi; Hayashi, Hideki; Suzuki, Akio; Itoh, Yoshinori

    2018-04-20

    Because clinical data to confirm the safety and effectiveness of fosphenytoin, a prodrug of phenytoin, are insufficient, the length of administration of fosphenytoin is restricted. Nevertheless, some cases require fosphenytoin administration for more than a few days. The aim of this study was to retrospectively investigate the serum concentration of phenytoin in adult Japanese patients who received intravenous fosphenytoin therapy for more than 3 days. Patients injected with intravenous fosphenytoin for more than 3 days at Gifu University Hospital between January 2012 and September 2014 were enrolled. Individual pharmacokinetic parameters were predicted by Bayesian estimation using NONMEM software, and the maintenance dose of fosphenytoin required to maintain the therapeutic trough concentration (10-20 μg/mL) was calculated from the parameters. Among a total of 8 patients, the serum trough concentration of phenytoin decreased with each day after repeated injection of fosphenytoin. The incidence rate of significant convulsive seizures was increased time-dependently (0% on day 1, 12.5% on day 2, 25% on day 3, and 66.7% on day 4 and after). Phenytoin clearance showed a time-dependent increase. The maintenance dose of fosphenytoin required to maintain the therapeutic trough concentration was simulated to be 779.8 ± 316.8 mg/day, a dose that was markedly higher than the actual maintenance dose (414.1 ± 55.7 mg/day). Prolonged use of fosphenytoin for such patients as those with autoimmune-mediated encephalopathy accompanied with reflux disease and/or ileus time-dependently decreased the serum concentration of phenytoin and increased the risk of convulsion. Therefore, the maintenance dose should be increased to maintain the therapeutic serum concentration.

  5. Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

    Directory of Open Access Journals (Sweden)

    Christine Laura Dixon

    2015-12-01

    Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

  6. Species dependence of [{sup 64}Cu]Cu-Bis(thiosemicarbazone) radiopharmaceutical binding to serum albumins

    Energy Technology Data Exchange (ETDEWEB)

    Basken, Nathan E. [Division of Nuclear Pharmacy, Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907 (United States)], E-mail: nbasken@purdue.edu; Mathias, Carla J. [Division of Nuclear Pharmacy, Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907 (United States); Lipka, Alexander E. [Department of Statistics, Purdue University, West Lafayette, IN 47907 (United States); Green, Mark A. [Division of Nuclear Pharmacy, Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907 (United States)], E-mail: magreen@purdue.edu

    2008-04-15

    Introduction: Interactions of three copper(II) bis(thiosemicarbazone) positron emission tomography radiopharmaceuticals with human serum albumin, and the serum albumins of four additional mammalian species, were evaluated. Methods: {sup 64}Cu-labeled diacetyl bis(N{sup 4}-methylthiosemicarbazonato)copper(II) (Cu-ATSM), pyruvaldehyde bis(N{sup 4}-methylthiosemicarbazonato)copper(II) (Cu-PTSM) and ethylglyoxal bis(thiosemicarbazonato)copper(II) (Cu-ETS) were synthesized and their binding to human, canine, rat, baboon and porcine serum albumins quantified by ultrafiltration. Protein binding was also measured for each tracer in human, porcine, rat and mouse serum. Results: The interaction of these neutral, lipophilic copper chelates with serum albumin is highly compound- and species-dependent. Cu-PTSM and Cu-ATSM exhibit particularly high affinity for human serum albumin (HSA), while the albumin binding of Cu-ETS is relatively insensitive to species. At HSA concentrations of 40 mg/ml, '% free' (non-albumin-bound) levels of radiopharmaceutical were 4.0{+-}0.1%, 5.3{+-}0.2% and 38.6{+-}0.8% for Cu-PTSM, Cu-ATSM and Cu-ETS, respectively. Conclusions: Species-dependent variations in radiopharmaceutical binding to serum albumin may need to be considered when using animal models to predict the distribution and kinetics of these compounds in humans.

  7. Comparative study on 2,2′,4,5,5′-pentachlorobiphenyl-mediated decrease in serum thyroxine level between C57BL/6 and its transthyretin-deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Yoshihisa, E-mail: kato@kph.bunri-u.ac.jp [Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Kagawa 769-2193 (Japan); Tamaki, Sekihiro [School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526 (Japan); Haraguchi, Koichi [Daiichi College of Pharmaceutical Sciences, Fukuoka 815-8511 (Japan); Ikushiro, Shin-ichi [Faculty of Engineering, Toyama Prefectural University, Toyama 939-0398 (Japan); Sekimoto, Masashi [School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526 (Japan); Ohta, Chiho [Faculty of Nutritional Sciences, Nakamura Gakuen University, Fukuoka 814-0198 (Japan); Endo, Tetsuya [Faculty of Pharmaceutical Sciences, Health Science University of Hokkaido, Hokkaido 061-0293 (Japan); Koga, Nobuyuki [Faculty of Nutritional Sciences, Nakamura Gakuen University, Fukuoka 814-0198 (Japan); Yamada, Shizuo; Degawa, Masakuni [School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526 (Japan)

    2012-09-15

    The relationships between the changes in the levels of serum total thyroxine (T{sub 4}), serum T{sub 4}-transthyretin (TTR) complex, and accumulation of T{sub 4} in tissues by 2,2′,4,5,5′-pentachlorobiphenyl (PentaCB) were examined using wild-type C57BL/6 (WT) and its TTR-deficient (TTR-null) mice. The constitutive level of serum total T{sub 4} was much higher in WT mice than in TTR-null mice. In WT mice 4 days after a single intraperitoneal injection with PentaCB (112 mg/kg), serum total T{sub 4} level was significantly decreased along with a decrease in serum T{sub 4}–TTR complex, and the levels of serum total T{sub 4} in the PentaCB-treated WT mice were almost the same to those in PentaCB-untreated (control) TTR-null mice. In addition, a slight decrease in serum total T{sub 4} by PentaCB treatment was observed in TTR-null mice. Furthermore, clearance of [{sup 125}I]T{sub 4} from the serum after [{sup 125}I]T{sub 4}-administration was promoted by the PentaCB-pretreatment in either strain of mice, especially WT mice. On the other hand, accumulation level of [{sup 125}I]T{sub 4} in the liver, but not in extrahepatic tissues, was strikingly enhanced in the PentaCB-pretreated WT and TTR-null mice. Furthermore, in both strains of mice, PentaCB-pretreatment led to significant increases in the steady-state distribution volume of [{sup 125}I]T{sub 4} and the concentration ratio of the liver to serum. The present findings demonstrate that PentaCB-mediated decrease in serum T{sub 4} level occurs mainly through increase in accumulation level of T{sub 4} in the liver and further indicate that the increased accumulation of T{sub 4} in the liver of WT mice is primarily dependent on the PentaCB-mediated inhibition of serum T{sub 4}–TTR complex formation.

  8. Decreased serum level of NGF in alcohol-dependent patients with declined executive function

    Directory of Open Access Journals (Sweden)

    Bae H

    2014-11-01

    Full Text Available Hwallip Bae,1 Youngsun Ra,1 Changwoo Han,2 Dai-Jin Kim3 1Department of Psychiatry, Myongji Hospital, Goyang, 2Department of Psychiatry, Keyo Hospital, Uiwang, 3Department of Psychiatry, Seoul St Mary’s Hospital, College of Medicine, Catholic University of Korea, Seoul, South Korea Abstract: The role of neurotrophic factors has been highlighted as a cause of decline in the cognitive function of alcohol-dependent patients. It is known that nerve-growth factor (NGF, one of the neurotrophins, is related to the growth and differentiation of nerve cells, as well as to a decline in cognitive function. The purpose of this study was to investigate the relationship between decreased NGF levels and cognitive decline in alcohol-dependent patients. The serum concentration of NGF was measured in 38 patients with chronic alcohol dependence, and several neuropsychological tests were also performed for cognitive function assessment. The results indicated a significant correlation between serum NGF level and the trail-making test part B, which evaluates executive function, but did not show a significant correlation with other cognitive function tests. An increased serum level of NGF was associated with a decreased completion time in the trail-making test B, and this finding indicates that a high serum level of NGF is related to greater executive function. This finding may imply a protective role of NGF in preventing neuron damage among patients with alcohol dependence. Larger controlled studies will be necessary in the future to investigate this issue further. Keywords: nerve-growth factor, alcohol dependence, executive function, trail-making test

  9. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    Science.gov (United States)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  10. Zolpidem reduces the blood oxygen level-dependent signal during visual system stimulation.

    Science.gov (United States)

    Licata, Stephanie C; Lowen, Steven B; Trksak, George H; Maclean, Robert R; Lukas, Scott E

    2011-08-15

    Zolpidem is a short-acting imidazopyridine hypnotic that binds at the benzodiazepine binding site on specific GABA(A) receptors to enhance fast inhibitory neurotransmission. The behavioral and receptor pharmacology of zolpidem has been studied extensively, but little is known about its neuronal substrates in vivo. In the present within-subject, double-blind, and placebo-controlled study, blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) at 3 Tesla was used to assess the effects of zolpidem within the brain. Healthy participants (n=12) were scanned 60 min after acute oral administration of zolpidem (0, 5, 10, or 20mg), and changes in BOLD signal were measured in the visual cortex during presentation of a flashing checkerboard. Heart rate and oxygen saturation were monitored continuously throughout the session. Zolpidem (10 and 20mg) reduced the robust visual system activation produced by presentation of this stimulus, but had no effects on physiological activity during the fMRI scan. Zolpidem's modulation of the BOLD signal within the visual cortex is consistent with the abundant distribution of GABA(A) receptors localized in this region, as well as previous studies showing a relationship between increased GABA-mediated neuronal inhibition and a reduction in BOLD activation. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Zolpidem reduces the blood oxygen level-dependent signal during visual system stimulation

    Science.gov (United States)

    Licata, Stephanie C.; Lowen, Steven B.; Trksak, George H.; MacLean, Robert R.; Lukas, Scott E.

    2011-01-01

    Zolpidem is a short-acting imidazopyridine hypnotic that binds at the benzodiazepine binding site on specific GABAA receptors to enhance fast inhibitory neurotransmission. The behavioral and receptor pharmacology of zolpidem has been studied extensively, but little is known about its neuronal substrates in vivo. In the present within-subject, double-blind, and placebo-controlled study, blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) at 3 Tesla was used to assess the effects of zolpidem within the brain. Healthy participants (n=12) were scanned 60 minutes after acute oral administration of zolpidem (0, 5, 10, or 20 mg), and changes in BOLD signal were measured in the visual cortex during presentation of a flashing checkerboard. Heart rate and oxygen saturation were monitored continuously throughout the session. Zolpidem (10 and 20 mg) reduced the robust visual system activation produced by presentation of this stimulus, but had no effects on physiological activity during the fMRI scan. Zolpidem’s modulation of the BOLD signal within the visual cortex is consistent with the abundant distribution of GABAA receptors localized in this region, as well as previous studies showing a relationship between increased GABA-mediated neuronal inhibition and a reduction in BOLD activation. PMID:21640782

  12. Exercisers' identities and exercise dependence: the mediating effect of exercise commitment.

    Science.gov (United States)

    Lu, Frank Jing-Horng; Hsu, Eva Ya-Wen; Wang, Junn-Ming; Huang, Mei-Yao; Chang, Jo-Ning; Wang, Chien-Hsin

    2012-10-01

    The purpose of this study was to examine the associations of exercise identity, exercise commitment, exercise dependence, and, particularly, the mediating effects of exercise commitment on the relationship between exercise identity and exercise dependence. 253 Taiwanese regular exercisers completed measures, including the Exercise Dependence Scale-Revised, the Exercise Identity Scale, the Exercise Commitment Scale, and the Godin Leisure Time Exercise Questionnaire. Results showed that exercise identity, exercise dependence, and two types of exercise commitment were moderately to highly correlated. Furthermore, structural equation modelling indicated that a "have to" commitment partially mediated the relationship between exercise identity and exercise dependence. Based on the mediating role of a "have to" commitment, the findings are particularly informative to exercise instructors and for exercise program managers.

  13. Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway.

    Science.gov (United States)

    Lim, Jung Hwa; Jung, Cho-Rok; Lee, Chan-Hee; Im, Dong-Soo

    2008-11-01

    E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.

  14. PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

    Science.gov (United States)

    Li, Li; Zhao, Lei; Wang, Yang; Ma, Ke-tao; Shi, Wen-yan; Wang, Ying-zi; Si, Jun-qiang

    2015-02-01

    The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

  15. Maternal Separation during Breastfeeding Induces Gender-Dependent Changes in Anxiety and the GABA-A Receptor Alpha-Subunit in Adult Wistar Rats.

    Directory of Open Access Journals (Sweden)

    Diego Armando León Rodríguez

    Full Text Available Different models of rodent maternal separation (MS have been used to investigate long-term neurobiological and behavioral changes, associated with early stress. However, few studies have involved the analysis of sex-related differences in central anxiety modulation. This study investigated whether MS during breastfeeding affected adult males and females in terms of anxiety and brain GABA-A receptor-alpha-subunit immunoreactivity. The brain areas analyzed were the amygdale (AM, hippocampus (HP, medial prefrontal cortex (mPFC, medial preoptic area (POA and paraventricular nucleus (PVN. Rats were housed under a reversed light/dark cycle (lights off at 7∶00 h with access to water and food ad libitum. Animals underwent MS twice daily during the dark cycle from postnatal day 1 to postnatal day 21. Behavior was tested when rats were 65-70 days old using the elevated plus maze and after brains were treated for immunohistochemistry. We found that separated females spent more time in the open arms and showed more head dipping behavior compared with controls. The separated males spent more time in the center of the maze and engaged in more stretching behavior than the controls. Immunohistochemistry showed that separated females had less immunostained cells in the HP, mPFC, PVN and POA, while separated males had fewer immunolabeled cells in the PFC, PVN and AM. These results could indicate that MS has gender-specific effects on anxiety behaviors and that these effects are likely related to developmental alterations involving GABA-A neurotransmission.

  16. Ventral tegmental area GABA neurons and opiate motivation

    Science.gov (United States)

    Vargas-Perez, Hector; Mabey, Jennifer K.; Shin, Samuel I.; Steffensen, Scott C.; van der Kooy, Derek

    2013-01-01

    Rational Past research has demonstrated that when an animal changes from a previously drug-naive to an opiate-dependent and withdrawn state, morphine’s motivational effects are switched from a tegmental pedunculopontine nucleus (TPP)-dependent to a dopamine-dependent pathway. Interestingly, a corresponding change is observed in ventral tegmental area (VTA) GABAA receptors, which change from mediating hyperpolarization of VTA GABA neurons to mediating depolarization. Objectives The present study investigated whether pharmacological manipulation of VTA GABAA receptor activity could directly influence the mechanisms underlying opiate motivation. Results Using an unbiased place conditioning procedure, we demonstrated that in Wistar rats, intra-VTA administration of furosemide, a Cl− cotransporter inhibitor, was able to promote a switch in the mechanisms underlying morphine’s motivational properties, one which is normally observed only after chronic opiate exposure. This behavioral switch was prevented by intra-VTA administration of acetazolamide, an inhibitor of the bicarbonate ion-producing carbonic anhydrase enzyme. Electrophysiological recordings of mouse VTA showed that furosemide reduced the sensitivity of VTA GABA neurons to inhibition by the GABAA receptor agonist muscimol, instead increasing the firing rate of a significant subset of these GABA neurons. Conclusion Our results suggest that the carbonic anhydrase enzyme may constitute part of a common VTA GABA neuron-based biological pathway responsible for controlling the mechanisms underlying opiate motivation, supporting the hypothesis that VTA GABAA receptor hyperpolarization or depolarization is responsible for selecting TPP- or dopamine-dependent motivational outputs, respectively. PMID:23392354

  17. A rapid microtiter plate serum bactericidal assay method for determining serum complement-mediated killing of Mannheimia haemolytica.

    Science.gov (United States)

    Ayalew, Sahlu; Confer, Anthony W; Shrestha, Binu; Payton, Mark E

    2012-05-01

    In this study, we describe a rapid microtiter serum bactericidal assay (RMSBA) that can be used to measure the functionality of immune sera. It quantifies bactericidal activity of immune sera in the presence of complement against a homologous bacterium, M. haemolytica in this case. There is high correlation between data from RMSBA and standard complement-mediated bacterial killing assay (r=0.756; p<0.0001). The RMSBA activity of sera can be generated in less than 5 h instead of overnight incubation. RMSBA costs substantially less in terms of time, labor, and resources and is highly reproducible. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Differential Potency of 2,6-Dimethylcyclohexanol Isomers for Positive Modulation of GABAA Receptor Currents.

    Science.gov (United States)

    Chowdhury, Luvana; Croft, Celine J; Goel, Shikha; Zaman, Naina; Tai, Angela C-S; Walch, Erin M; Smith, Kelly; Page, Alexandra; Shea, Kevin M; Hall, C Dennis; Jishkariani, D; Pillai, Girinath G; Hall, Adam C

    2016-06-01

    GABAA receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6-dimethylcyclohexanol diastereomers were investigated on human GABAA (α1β3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulation of submaximal GABA responses. For example, the addition of 30 μM cis,cis-isomer resulted in an approximately 2- to 3-fold enhancement of the EC20 GABA current. Coapplications of 30 μM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > > cis,trans-isomer. In molecular modeling studies, the three cyclohexanol isomers bound with the highest binding energies to a pocket within transmembrane helices M1 and M2 of the β3 subunit through hydrogen-bonding interactions with a glutamine at the 224 position and a tyrosine at the 220 position. The energies for binding to and hydrogen-bond lengths within this pocket corresponded with the relative potencies of the agents for positive modulation of GABAA receptor currents (cis,cis > trans,trans > cis,trans-2,6-dimethylcyclohexanol). In conclusion, the stereochemical configuration within the dimethylcyclohexanols is an important molecular feature in conferring positive modulation of GABAA receptor activity and for binding to the receptor, a consideration that needs to be taken into account when designing novel anesthetics with enhanced therapeutic indices. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Serum-dependent expression of promyelocytic leukemia protein suppresses propagation of influenza virus

    International Nuclear Information System (INIS)

    Iki, Shigeo; Yokota, Shin-ichi; Okabayashi, Tamaki; Yokosawa, Noriko; Nagata, Kyosuke; Fujii, Nobuhiro

    2005-01-01

    The rate of propagation of influenza virus in human adenocarcinoma Caco-2 cells was found to negatively correlate with the concentration of fetal bovine serum (FBS) in the culture medium. Virus replicated more rapidly at lower FBS concentrations (0 or 2%) than at higher concentrations (10 or 20%) during an early stage of infection. Basal and interferon (IFN)-induced levels of typical IFN-inducible anti-viral proteins, such as 2',5'-oligoadenylate synthetase, dsRNA-activated protein kinase and MxA, were unaffected by variation in FBS concentrations. But promyelocytic leukemia protein (PML) was expressed in a serum-dependent manner. In particular, the 65 to 70 kDa isoform of PML was markedly upregulated following the addition of serum. In contrast, other isoforms were induced by IFN treatment, and weakly induced by FBS concentrations. Immunofluorescence microscopy indicated that PML was mainly formed nuclear bodies in Caco-2 cells at various FBS concentrations, and the levels of the PML-nuclear bodies were upregulated by FBS. Overexpression of PML isoform consisting of 560 or 633 amino acid residues by transfection of expression plasmid results in significantly delayed viral replication rate in Caco-2 cells. On the other hand, downregulation of PML expression by RNAi enhanced viral replication. These results indicate that PML isoforms which are expressed in a serum-dependent manner suppress the propagation of influenza virus at an early stage of infection

  20. Recruitment of GABA(A) receptors and fearfulness in chicks: modulation by systemic insulin and/or epinephrine.

    Science.gov (United States)

    Cid, Mariana Paula; Toledo, Carolina Maribel; Salvatierra, Nancy Alicia

    2013-02-01

    One-day-old chicks were individually assessed on their latency to peck pebbles, and categorized as low latency (LL) or high latency (HL) according to fear. Interactions between acute stress and systemic insulin and epinephrine on GABA(A) receptor density in the forebrain were studied. At 10 days of life, LL and HL chicks were intraperitoneally injected with insulin, epinephrine or saline, and immediately after stressed by partial water immersion for 15 min and killed by decapitation. Forebrains were dissected and the GABA(A) receptor density was measured ex vivo by the (3)[H]-flunitrazepam binding assay in synaptosomes. In non-stressed chicks, insulin (non-hypoglycemic dose) at 2.50 IU/kg of body weight incremented the Bmax by 40.53% in the HL chicks compared to saline group whereas no significant differences were observed between individuals in the LL subpopulation. Additionally, insulin increased the Bmax (23.48%) in the HL group with respect to the LL ones, indicating that the insulin responses were different according to the anxiety of each category. Epinephrine administration (0.25 and 0.50mg/kg) incremented the Bmax in non-stressed chicks, in the LL group by about 37% and 33%, respectively, compared to ones injected with saline. In the stressed chicks, 0.25mg/kg bw epinephrine increased the Bmax significantly in the HL group by about 24% compared to saline, suggesting that the effect of epinephrine was only observed in the HL group under acute stress conditions. Similarly, the same epinephrine doses co-administered with insulin increased the receptor density in both subpopulations and also showed that the highest dose of epinephrine did not further increase the maximum density of GABA(A)R in HL chicks. These results suggest that systemic epinephrine, perhaps by evoking central norepinephrine release, modulated the increase in the forebrain GABA(A) receptor recruitment induced by both insulin and stress in different ways depending on the subpopulation

  1. Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

    Directory of Open Access Journals (Sweden)

    M.R. Hespanhol

    2002-03-01

    Full Text Available Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18 and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA by mouse peritoneal macrophages. We observed that a macrophages are able to recognize (bind to these red cells, b this interaction can be inhibited by denatured BSA in the fluid phase, c there is no phagocytosis of these particles by normal macrophages, d phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A.

  2. Basal serum pancreatic polypeptide is dependent on age and gender in an adult population

    DEFF Research Database (Denmark)

    Brimnes Damholt, M; Rasmussen, B K; Hilsted, L

    1997-01-01

    This study is the first epidemiologically based study of basal levels of serum pancreatic polypeptide (s-PP). The basal level of serum PP has become a field of interest mainly due to the role of PP as an endocrine tumour marker, and as a marker of pancreatic neuroendocrine function after pancreas...... a monospecific radioimmunoassay. Fasting serum pancreatic polypeptide depended on age and gender. The results demonstrated that fasting pancreatic polypeptide levels increase exponentially with age. Fitted separately for each sex, basal serum pancreatic polypeptide was found to increase by approximately 3% per...... reports on the fasting levels of serum pancreatic polypeptide are most likely due to lack of adjustment for age and gender. Thus, variation due to age and gender should be considered in evaluating fasting levels of serum pancreatic polypeptide. Whether similar considerations are important when evaluating...

  3. The effect of the GABA-A agonist THIP on regional cortical blood flow in humans. A new test of hemispheric dominance

    DEFF Research Database (Denmark)

    Roland, PE; Friberg, L

    1988-01-01

    We studied the effect of a gamma-aminobutyric acid (GABA)-A receptor-induced postsynaptic inhibition on regional CBF (rCBF) in awake humans. For this purpose we used a new specific GABA-A agonist, 4,5,6,7-tetrahydroisoxazolo(5,4)-pyridin-3-ol (THIP). As part of a new diagnostic procedure for the ...

  4. GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

    Science.gov (United States)

    Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth; Johnson, David; Borghese, Cecilia M.; Hanrahan, Jane R.; Johnston, Graham A. R.; Chebib, Mary; Harris, R. Adron

    2014-01-01

    GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo. PMID:24454882

  5. GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

    Directory of Open Access Journals (Sweden)

    Yuri A Blednov

    Full Text Available GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR, and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ1" antagonist, when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests, but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ2" antagonist did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

  6. GABA(A) receptor- and GABA transporter polymorphisms and risk for essential tremor

    DEFF Research Database (Denmark)

    Thier, S; Kuhlenbäumer, G; Lorenz, D

    2011-01-01

    Background:  Clinical features and animal models of essential tremor (ET) suggest gamma-aminobutyric acid A receptor (GABA(A) R) subunits and GABA transporters as putative candidate genes. Methods:  A total of 503 ET cases and 818 controls were investigated for an association between polymorphisms...

  7. Dose and duration dependent of aluminium in the serum liver and ...

    African Journals Online (AJOL)

    An atomic absorption spectrophotometric analysis on dose and duration dependent aluminum concentration in serum, liver and brain digests of three groups of male Wistar albino rats were investigated after seven and fourteen days of daily 0.38mg/kg, 3.8mg/kg and 38mg/kg aluminum administration respectively.

  8. Age-Dependent Decrease in Serum Transforming Growth Factor (TGF-Beta 1 in Healthy Japanese Individuals; Population Study of Serum TGF-Beta 1 Level in Japanese

    Directory of Open Access Journals (Sweden)

    Yoshihiro Okamoto

    2005-01-01

    Full Text Available Transforming growth factor-beta1 (TGF-β1, a multi-functional cytokine, is involved in regulating a variety of cellular activities and the serum/plasma TGF-β1 level is altered with various diseases. However, most published reports have described adult patients, and so we investigated the clinical significance of serum TGF-β1 level in pediatric patients. The diagnostic application of the measurement of serum TGF-β1 level depends critically on the control value, however, there is no information on the control value of serum TGF-β1 for children.

  9. γ-glutamyl transpeptidase 1 specifically suppresses green-light avoidance via GABAA receptors in Drosophila.

    Science.gov (United States)

    Liu, Jiangqu; Gong, Zhefeng; Liu, Li

    2014-08-01

    Drosophila larvae innately show light avoidance behavior. Compared with robust blue-light avoidance, larvae exhibit relatively weaker green-light responses. In our previous screening for genes involved in larval light avoidance, compared with control w(1118) larvae, larvae with γ-glutamyl transpeptidase 1 (Ggt-1) knockdown or Ggt-1 mutation were found to exhibit higher percentage of green-light avoidance which was mediated by Rhodopsin6 (Rh6) photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt-1 in different tissues, we found that Ggt-1 in malpighian tubules was both necessary and sufficient for green-light avoidance. Our results showed that glutamate levels were lower in Ggt-1 null mutants compared with controls. Feeding Ggt-1 null mutants glutamate can normalize green-light avoidance, indicating that high glutamate concentrations suppressed larval green-light avoidance. However, rather than directly, glutamate affected green-light avoidance indirectly through GABA, the level of which was also lower in Ggt-1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABAA receptor, both exhibit elevated levels of green-light avoidance. Thus, our results elucidate the neurobiological mechanisms mediating green-light avoidance, which was inhibited in wild-type larvae. © 2014 International Society for Neurochemistry.

  10. The role of GABA in NMDA-dependent long term depression (LTD) of rat medial vestibular nuclei.

    Science.gov (United States)

    Grassi, S; Della Torre, G; Capocchi, G; Zampolini, M; Pettorossi, V E

    1995-11-20

    The role of GABA in NMDA-dependent long term depression (LTD) in the medial vestibular nuclei (MVN) was studied on rat brainstem slices. High frequency stimulation (HFS) of the primary vestibular afferents induces a long lasting reduction of the polysynaptic (N2) component of the field potentials recorded in the dorsal portion of the MVN. The induction but not the maintenance of this depression was abolished by AP5, a specific blocking agent for glutamate NMDA receptors. The involvement of GABA in mediating the depression was checked by applying the GABAA and GABAB receptor antagonists, bicuculline and saclofen, before and after HFS. Under bicuculline and saclofen perfusion, HFS provoked a slight potentiation of the N2 wave, while the N2 depression clearly emerged after drug wash-out. This indicates that GABA is not involved in inducing the long term effect, but it is necessary for its expression. Similarly, the LTD reversed and a slight potentiation appeared when both drugs were administered after its induction. Most of these effects were due to the bicuculline, suggesting that GABAA receptors contribute to LTD more than GABAB do. According to our results, it is unlikely that the long lasting vestibular depression is the result of a homosynaptic LTD. On the contrary, our findings suggest that the depression is due to an enhancement of the GABA inhibitory effect, caused by an HFS dependent increase in gabaergic interneuron activity, which resets vestibular neuron excitability at a lower level.

  11. Modulation of the cough reflex by GABAA receptors in the caudal ventral respiratory group of the rabbit

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    Elenia eCinelli

    2012-10-01

    Full Text Available We have previously shown that the caudal ventral respiratory group (cVRG is a possible site of action of some antitussive drugs and plays a crucial role in determining both the expiratory and inspiratory components of the cough motor pattern. In addition, it has been reported that medullary expiratory neurons of the cVRG are subject to potent GABAergic gain modulation. This study was devoted to investigate the role of cVRG GABAA receptors in the control of baseline respiratory activity and cough responses to mechanical and chemical (citric acid stimulation of the tracheobronchial tree. To this purpose, bilateral microinjections (30-50 nl of bicuculline or muscimol were performed into the cVRG of pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bicuculline (1 mM increased peak abdominal activity and respiratory frequency due to decreases in TE. Cough responses were potentiated mainly owing to increases in the cough number. The recovery was observed within ~ 2 h. On the contrary, muscimol (0.3 mM abolished abdominal activity and decreased respiratory frequency due to increases in TE. In addition, cough responses were progressively reduced and completely suppressed within ~ 20 min. Partial recovery of cough responses was achieved after ~ 3 h or within ~ 5 min following bicuculline microinjections at the same locations. The sneeze reflex induced by mechanical stimulation of the nasal mucosa persisted following bicuculline and muscimol microinjections. However, the number and intensity of expiratory thrusts were enhanced by bicuculline and suppressed by muscimol. The results provide evidence that a potent GABAA-mediated inhibitory modulation is exerted at the level of the cVRG not only on respiratory activity, but also on cough and sneeze reflex responses.

  12. Positive selection within the Schizophrenia-associated GABA(A receptor beta(2 gene.

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    Wing-Sze Lo

    Full Text Available The gamma-aminobutyric acid type-A (GABA(A receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABA(A receptor beta(2 subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced beta(2 isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of beta(2, especially its long isoform. Electrophysiological analysis showed that this long beta(2 isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABA(A receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.

  13. Targeted deletion of the GABRA2 gene encoding alpha2-subunits of GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates.

    Science.gov (United States)

    Dixon, C I; Rosahl, T W; Stephens, D N

    2008-07-01

    Mice with point-mutated alpha2 GABA(A) receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in the conditioned emotional response (CER) test, but show normal anxiolytic effects of a barbiturate. We investigated the consequence of deleting the alpha2-subunit on acquisition of the CER with increasing intensity of footshock, and on the anxiolytic efficacy of a benzodiazepine, diazepam, and a barbiturate, pentobarbital. alpha2 knockout (KO) and wildtype (WT) mice were trained in a conditioned emotional response (CER) task, in which lever pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a compound light/tone conditioned stimulus (CS+) that predicted footshock. The ability of diazepam and of pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. There were no differences between the genotypes in shock sensitivity, as assessed by their flinch responses to increasing levels of shock. However, alpha2 KO mice showed a greater suppression of lever pressing than WT littermates in the presence of a compound cue signalling footshock. Diazepam (0, 0.5, 1 and 2 mg/kg) induced a dose-dependent anxiolytic-like effect in WT mice but no such effect was seen in KO mice. Similarly, although pentobarbital (20 mg/kg) reduced the ability of the CS+ to reduce lever pressing rates in WT mice, this effect was not seen in the KO. These findings suggest that alpha2-containing GABA(A) receptors mediate the anxiolytic effects of barbiturates, as well as benzodiazepines, and that they may be involved in neuronal circuits underlying conditioned anxiety.

  14. Block of GABA(A) receptor ion channel by penicillin: electrophysiological and modeling insights toward the mechanism.

    Science.gov (United States)

    Rossokhin, Alexey V; Sharonova, Irina N; Bukanova, Julia V; Kolbaev, Sergey N; Skrebitsky, Vladimir G

    2014-11-01

    GABA(A) receptors (GABA(A)R) mainly mediate fast inhibitory neurotransmission in the central nervous system. Different classes of modulators target GABA(A)R properties. Penicillin G (PNG) belongs to the class of noncompetitive antagonists blocking the open GABA(A)R and is a prototype of β-lactam antibiotics. In this study, we combined electrophysiological and modeling approaches to investigate the peculiarities of PNG blockade of GABA-activated currents recorded from isolated rat Purkinje cells and to predict the PNG binding site. Whole-cell patch-сlamp recording and fast application system was used in the electrophysiological experiments. PNG block developed after channel activation and increased with membrane depolarization suggesting that the ligand binds within the open channel pore. PNG blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC50 value of 1.12mM at -70mV. The termination of GABA and PNG co-application was followed by a transient tail current. Protection of the tail current from bicuculline block and dependence of its kinetic parameters on agonist affinity suggest that PNG acts as a sequential open channel blocker that prevents agonist dissociation while the channel remains blocked. We built the GABA(A)R models based on nAChR and GLIC structures and performed an unbiased systematic search of the PNG binding site. Monte-Carlo energy minimization was used to find the lowest energy binding modes. We have shown that PNG binds close to the intracellular vestibule. In both models the maximum contribution to the energy of ligand-receptor interactions revealed residues located on the level of 2', 6' and 9' rings formed by a bundle of M2 transmembrane segments, indicating that these residues most likely participate in PNG binding. The predicted structural models support the described mechanism of PNG block. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Activation of GABA(A) receptors in the accessory olfactory bulb does not prevent the formation of an olfactory memory in mice.

    Science.gov (United States)

    Otsuka, T; Hashida, M; Oka, T; Kaba, H

    2001-07-01

    When female mice are mated, they form a memory to the pheromonal signal of their male partner. The neural mechanisms underlying this memory involve changes at the reciprocal dendrodendritic synapses between glutamatergic mitral cells and gamma-aminobutyric acid (GABA)-ergic granule cells in the accessory olfactory bulb (AOB). Blockade of GABA(A) receptors in the AOB leads to the formation of an olfactory memory. In an attempt to disrupt memory formation at mating, we used local infusions of the GABA(A) receptor agonist muscimol into the AOB during the critical period for memory formation. Muscimol across a wide range of doses (1-1000 pmol) did not prevent memory formation. The resistance of this memory to GABA(A) receptor activation may reflect the complexity of synaptic microcircuits in the AOB.

  16. Inositol polyphosphate multikinase is a coactivator for serum response factor-dependent induction of immediate early genes

    Science.gov (United States)

    Kim, Eunha; Tyagi, Richa; Lee, Joo-Young; Park, Jina; Kim, Young-ran; Beon, Jiyoon; Chen, Po Yu; Cha, Jiyoung Y.; Snyder, Solomon H.; Kim, Seyun

    2013-01-01

    Inositol polyphosphate multikinase (IPMK) is a notably pleiotropic protein. It displays both inositol phosphate kinase and phosphatidylinositol kinase catalytic activities. Noncatalytically, IPMK stabilizes the mammalian target of rapamycin complex 1 and acts as a transcriptional coactivator for CREB-binding protein/E1A binding protein p300 and tumor suppressor protein p53. Serum response factor (SRF) is a major transcription factor for a wide range of immediate early genes. We report that IPMK, in a noncatalytic role, is a transcriptional coactivator for SRF mediating the transcription of immediate early genes. Stimulation by serum of many immediate early genes is greatly reduced by IPMK deletion. IPMK stimulates expression of these genes, an influence also displayed by catalytically inactive IPMK. IPMK acts by binding directly to SRF and thereby enhancing interactions of SRF with the serum response element of diverse genes. PMID:24248338

  17. Insulin reduces neuronal excitability by turning on GABA(A channels that generate tonic current.

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    Zhe Jin

    Full Text Available Insulin signaling to the brain is important not only for metabolic homeostasis but also for higher brain functions such as cognition. GABA (γ-aminobutyric acid decreases neuronal excitability by activating GABA(A channels that generate phasic and tonic currents. The level of tonic inhibition in neurons varies. In the hippocampus, interneurons and dentate gyrus granule cells normally have significant tonic currents under basal conditions in contrast to the CA1 pyramidal neurons where it is minimal. Here we show in acute rat hippocampal slices that insulin (1 nM "turns on" new extrasynaptic GABA(A channels in CA1 pyramidal neurons resulting in decreased frequency of action potential firing. The channels are activated by more than million times lower GABA concentrations than synaptic channels, generate tonic currents and show outward rectification. The single-channel current amplitude is related to the GABA concentration resulting in a single-channel GABA affinity (EC(50 in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABA(A antagonists but have novel pharmacology as the benzodiazepine flumazenil and zolpidem are inverse agonists. The results show that tonic rather than synaptic conductances regulate basal neuronal excitability when significant tonic conductance is expressed and demonstrate an unexpected hormonal control of the inhibitory channel subtypes and excitability of hippocampal neurons. The insulin-induced new channels provide a specific target for rescuing cognition in health and disease.

  18. Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

    Science.gov (United States)

    Wu, Qian; Zhong, Zhao-Ming; Zhu, Si-Yuan; Liao, Cong-Rui; Pan, Ying; Zeng, Ji-Huan; Zheng, Shuai; Ding, Ruo-Ting; Lin, Qing-Song; Ye, Qing; Ye, Wen-Bin; Li, Wei; Chen, Jian-Ting

    2016-01-01

    Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

  19. Complement-mediated solubilization of immune complexes. Solubilization inhibition and complement factor levels in SLE patients

    DEFF Research Database (Denmark)

    Baatrup, Gunnar; Petersen, Ivan; Kappelgaard, E

    1984-01-01

    Thirty-two of 36 serum samples from 19 SLE patients showed reduced capacity to mediate complement-dependent solubilization of immune complexes (IC). SLE patients with nephritis exerted the lowest complement-mediated solubilization capacity (CMSC) whereas sera from patients with inactive disease g...

  20. In vitro blood-brain barrier permeability predictions for GABAA receptor modulating piperine analogs

    DEFF Research Database (Denmark)

    Eigenmann, Daniela Elisabeth; Dürig, Carmen; Jähne, Evelyn Andrea

    2016-01-01

    The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain by c...

  1. Lateral Orbitofrontal Cortical Modulation on the Medial Prefrontal Cortex-Amygdala Pathway: Differential Regulation of Intra-Amygdala GABAA and GABAB Receptors.

    Science.gov (United States)

    Chang, Chun-Hui

    2017-07-01

    The basolateral complex of the amygdala receives inputs from neocortical areas, including the medial prefrontal cortex and lateral orbitofrontal cortex. Earlier studies have shown that lateral orbitofrontal cortex activation exerts an inhibitory gating on medial prefrontal cortex-amygdala information flow. Here we examined the individual role of GABAA and GABAB receptors in this process. In vivo extracellular single-unit recordings were done in anesthetized rats. We searched amygdala neurons that fire in response to medial prefrontal cortex activation, tested lateral orbitofrontal cortex gating at different delays (lateral orbitofrontal cortex-medial prefrontal cortex delays: 25, 50, 100, 250, 500, and 1000 milliseconds), and examined differential contribution of GABAA and GABAB receptors with iontophoresis. Relative to baseline, lateral orbitofrontal cortex stimulation exerted an inhibitory modulatory gating on the medial prefrontal cortex-amygdala pathway and was effective up to a long delay of 500 ms (long-delay latencies at 100, 250, and 500 milliseconds). Moreover, blockade of intra-amygdala GABAA receptors with bicuculline abolished the lateral orbitofrontal cortex inhibitory gating at both short- (25 milliseconds) and long-delay (100 milliseconds) intervals, while blockade of GABAB receptors with saclofen reversed the inhibitory gating at long delay (100 milliseconds) only. Among the majority of the neurons examined (8 of 9), inactivation of either GABAA or GABAB receptors during baseline did not change evoked probability per se, suggesting that local feed-forward inhibitory mechanism is pathway specific. Our results suggest that the effect of lateral orbitofrontal cortex inhibitory modulatory gating was effective up to 500 milliseconds and that intra-amygdala GABAA and GABAB receptors differentially modulate the short- and long-delay lateral orbitofrontal cortex inhibitory gating on the medial prefrontal cortex-amygdala pathway. © The Author 2017

  2. GABA-A Receptor Modulation and Anticonvulsant, Anxiolytic, and Antidepressant Activities of Constituents from Artemisia indica Linn

    Directory of Open Access Journals (Sweden)

    Imran Khan

    2016-01-01

    Full Text Available Artemisia indica, also known as “Mugwort,” has been widely used in traditional medicines. However, few studies have investigated the effects of nonvolatile components of Artemisia indica on central nervous system’s function. Fractionation of Artemisia indica led to the isolation of carnosol, ursolic acid, and oleanolic acid which were evaluated for their effects on GABA-A receptors in electrophysiological studies in Xenopus oocytes and were subsequently investigated in mouse models of acute toxicity, convulsions (pentylenetetrazole induced seizures, depression (tail suspension and forced swim tests, and anxiety (elevated plus maze and light/dark box paradigms. Carnosol, ursolic acid, and oleanolic acid were found to be positive modulators of α1β2γ2L GABA-A receptors and the modulation was antagonized by flumazenil. Carnosol, ursolic acid, and oleanolic acid were found to be devoid of any signs of acute toxicity (50–200 mg/kg but elicited anticonvulsant, antidepressant, and anxiolytic activities. Thus carnosol, ursolic acid, and oleanolic acid demonstrated CNS activity in mouse models of anticonvulsant, antidepressant, and anxiolysis. The anxiolytic activity of all three compounds was ameliorated by flumazenil suggesting a mode of action via the benzodiazepine binding site of GABA-A receptors.

  3. CD40 dependent exacerbation of immune mediated hepatitis by hepatic CD11b+ Gr-1+ myeloid derived suppressor cells in tumor bearing mice

    Science.gov (United States)

    Kapanadze, Tamar; Medina-Echeverz, José; Gamrekelashvili, Jaba; Weiss, Jonathan M.; Wiltrout, Robert H.; Kapoor, Veena; Hawk, Nga; Terabe, Masaki; Berzofsky, Jay A.; Manns, Michael P.; Wang, Ena; Marincola, Francesco M.; Korangy, Firouzeh; Greten, Tim F.

    2015-01-01

    Immunosuppressive CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) accumulate in the livers of tumor-bearing mice. We studied hepatic MDSC in two murine models of immune mediated hepatitis. Unexpectedly, treatment of tumor bearing mice with Concanavalin A or α-Galactosylceramide resulted in increased ALT and AST serum levels in comparison to tumor free mice. Adoptive transfer of hepatic MDSC into naïve mice exacerbated Concanavalin A induced liver damage. Hepatic CD11b+Gr-1+ cells revealed a polarized pro-inflammatory gene signature after Concanavalin A treatment. An interferon gamma- dependent up-regulation of CD40 on hepatic CD11b+Gr-1+ cells along with an up-regulation of CD80, CD86, and CD1d after Concanavalin A treatment was observed. Concanavalin A treatment resulted in a loss of suppressor function by tumor-induced CD11b+Gr-1+ MDSC as well as enhanced reactive oxygen species-mediated hepatotoxicity. CD40 knockdown in hepatic MDSC led to increased arginase activity upon Concanavalin A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40−/− tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased reactive oxygen species production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSC act as pro-inflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner. PMID:25616156

  4. Subregion-specific modulation of excitatory input and dopaminergic output in the striatum by tonically activated glycine and GABAA receptors

    Directory of Open Access Journals (Sweden)

    Louise eAdermark

    2011-10-01

    Full Text Available The flow of cortical information through the basal ganglia is a complex spatiotemporal pattern of increased and decreased firing. The striatum is the biggest input nucleus to the basal ganglia and the aim of this study was to assess the role of inhibitory GABAA and glycine receptors in regulating synaptic activity in the dorsolateral (DLS and ventral striatum (nucleus accumbens, nAc. Local field potential recordings from coronal brain slices of juvenile and adult Wistar rats showed that GABAA receptors and strychnine-sensitive glycine receptors are tonically activated and inhibit excitatory input to the DLS and to the nAc. Strychnine-induced disinhibition of glutamatergic transmission was insensitive to the muscarinic receptor inhibitor scopolamine (10 µM, inhibited by the nicotinic acetylcholine receptor antagonist mecamylamine (10 µM and blocked by GABAA receptor inhibitors, suggesting that tonically activated glycine receptors depress excitatory input to the striatum through modulation of cholinergic and GABAergic neurotransmission. As an end-product example of striatal GABAergic output in vivo we measured dopamine release in the DLS and nAc by microdialysis in the awake and freely moving rat. Reversed dialysis of bicuculline (50 μM in perfusate only increased extrasynaptic dopamine levels in the nAc, while strychnine administered locally (200 μM in perfusate decreased dopamine output by 60% in both the DLS and nAc. Our data suggest that GABAA and glycine receptors are tonically activated and modulate striatal transmission in a partially sub-region specific manner.

  5. Sucrose/bovine serum albumin mediated biomimetic crystallization

    Indian Academy of Sciences (India)

    To understand the role of the sucrose/bovine serum albumin system in the biomineralization process, we have tested the influence of different concentration of the sucrose/bovine serum albumin (BSA) on calcium carbonate (CaCO3) precipitation. The CaCO3 crystals were characterized by scanning electron microscope ...

  6. Association of serum bicarbonate levels with mortality in patients with non-dialysis-dependent CKD

    Science.gov (United States)

    Kovesdy, Csaba P.; Anderson, John E.; Kalantar-Zadeh, Kamyar

    2009-01-01

    Background. Metabolic acidosis, usually manifested by low serum bicarbonate level, is common in chronic kidney disease (CKD) and appears to be associated with higher mortality in dialysis patients. It is not known whether a similar association is present in patients with non-dialysis-dependent CKD (NDD-CKD). Methods. We used multivariable-adjusted Cox models to examine the association between baseline and time-variable serum bicarbonate (measured as total CO2) with the outcomes of all-cause mortality and the composite of pre-dialysis mortality or end-stage renal disease in 1240 male patients with moderate and advanced NDD-CKD. Results. Serum bicarbonate showed a significant U-shaped association with all-cause mortality, with the highest mortality rate observed in patients with baseline serum bicarbonate levels <22 mmol/L [multivariable-adjusted hazard ratio (95% confidence interval) for patients with serum bicarbonate <22 mmol/L versus ≥22 mmol/L: 1.33 (1.05–1.69), P = 0.02] and the lowest mortality observed in patients with baseline serum bicarbonate of 26–29 mmol/L. The associations between lower serum bicarbonate level and mortality were more accentuated in subgroups of patients with better nutritional status and lower inflammation. Conclusions. Both lower and higher serum bicarbonates are associated with increased all-cause mortality in patients with moderate and advanced NDD-CKD. Clinical trials are needed to determine if therapeutic interventions aimed at optimizing serum bicarbonate can result in improved outcomes in this population. PMID:19015169

  7. Relative Neurotoxicity of Ivermectin and Moxidectin in Mdr1ab (−/−) Mice and Effects on Mammalian GABA(A) Channel Activity

    Science.gov (United States)

    Ménez, Cécile; Sutra, Jean-François; Prichard, Roger; Lespine, Anne

    2012-01-01

    The anthelmintics ivermectin (IVM) and moxidectin (MOX) display differences in toxicity in several host species. Entrance into the brain is restricted by the P-glycoprotein (P-gp) efflux transporter, while toxicity is mediated through the brain GABA(A) receptors. This study compared the toxicity of IVM and MOX in vivo and their interaction with GABA(A) receptors in vitro. Drug toxicity was assessed in Mdr1ab(−/−) mice P-gp-deficient after subcutaneous administration of increasing doses (0.11–2.0 and 0.23–12.9 µmol/kg for IVM and MOX in P-gp-deficient mice and half lethal doses (LD50) in wild-type mice). Survival was evaluated over 14-days. In Mdr1ab(−/−) mice, LD50 was 0.46 and 2.3 µmol/kg for IVM and MOX, respectively, demonstrating that MOX was less toxic than IVM. In P-gp-deficient mice, MOX had a lower brain-to-plasma concentration ratio and entered into the brain more slowly than IVM. The brain sublethal drug concentrations determined after administration of doses close to LD50 were, in Mdr1ab(−/−) and wild-type mice, respectively, 270 and 210 pmol/g for IVM and 830 and 740–1380 pmol/g for MOX, indicating that higher brain concentrations are required for MOX toxicity than IVM. In rat α1β2γ2 GABA channels expressed in Xenopus oocytes, IVM and MOX were both allosteric activators of the GABA-induced response. The Hill coefficient was 1.52±0.45 for IVM and 0.34±0.56 for MOX (p<0.001), while the maximum potentiation caused by IVM and MOX relative to GABA alone was 413.7±66.1 and 257.4±40.6%, respectively (p<0.05), showing that IVM causes a greater potentiation of GABA action on this receptor. Differences in the accumulation of IVM and MOX in the brain and in the interaction of IVM and MOX with GABA(A) receptors account for differences in neurotoxicity seen in intact and Mdr1-deficient animals. These differences in neurotoxicity of IVM and MOX are important in considering their use in humans. PMID:23133688

  8. Functional analysis of Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Hein, Estrid; Honoré, Christian Le Fèvre; Skjoedt, Mikkel-Ole

    2010-01-01

    assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition...... was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides...

  9. Concentration-dependent sedimentation properties of ferritin: implications for estimation of iron contents of serum ferritins

    International Nuclear Information System (INIS)

    Niitsu, Y.; Adachi, C.; Takahashi, F.; Goto, Y.; Kohgo, Y.; Urushizaki, I.; Listowsky, I.

    1985-01-01

    Serum ferritins from various sources sedimented at lower densities than tissue ferritins in sucrose gradient centrifugation systems. The sedimentation patterns of ferritins, however, were shown to be dependent on the concentration of the protein; as the concentration decreased the protein appeared to sediment at lower densities. Thus, at the low concentration levels usually used for analysis of serum ferritin, tissue ferritins also sedimented in the same lower density regions. Iron labeling experiments indicated that the sedimentation changes upon dilution were not due to release of iron or was there any indication that the protein dissociated into subunits. The anomalous sedimentation behavior of serum ferritin should therefore not be interpreted in terms of its iron content. The disclosure that serum ferritins may have full complements of iron is counter to the prevalent view that serum ferritins are low iron forms and has potential implications with regard to the sources and possible function of this protein in the circulation

  10. Effects of chronic infusion of a GABAA receptor agonist or antagonist into the vestibular nuclear complex on vestibular compensation in the guinea pig.

    Science.gov (United States)

    Gliddon, Catherine M; Darlington, Cynthia L; Smith, Paul F

    2005-06-01

    The aim of this study was to determine the effects of chronic infusion of a GABA(A) receptor agonist/antagonist into the ipsilateral or contralateral vestibular nuclear complex (VNC) on vestibular compensation, the process of behavioral recovery that occurs after unilateral vestibular deafferentation (UVD). This was achieved by a mini-osmotic pump that infused, over 30 h, muscimol or gabazine into the ipsilateral or contralateral VNC. Spontaneous nystagmus (SN), yaw head tilt (YHT), and roll head tilt (RHT) were measured. Infusion of muscimol or gabazine into either the ipsilateral or the contralateral VNC had little effect on SN compensation. In contrast, infusion of muscimol (250, 500, and 750 ng) into the contralateral VNC and gabazine (31.25, 62.5, and 125 ng) into the ipsilateral VNC significantly affected YHT and RHT (p 0.05). Interestingly, the effects of muscimol and gabazine on YHT and RHT were consistent throughout the first 30 h post-UVD. Infusion of muscimol (62.5, 125, and 250 ng) into the ipsilateral VNC and gabazine (125, 375, and 750 ng) into the contralateral VNC had little effect on YHT and RHT or their rate of compensation. These results suggest that the ipsilateral gabazine and contralateral muscimol infusions are modifying the expression of the symptoms without altering the mechanism of compensation. Furthermore, the neurochemical mechanism responsible for vestibular compensation can cope with the both the GABA(A) receptor-mediated and the UVD-induced decrease in resting activity.

  11. Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

    Science.gov (United States)

    Mellor, J R; Wisden, W; Randall, A D

    2000-07-10

    Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden

  12. Flavylium salts as in vitro precursors of potent ligands to brain GABA-A receptors

    DEFF Research Database (Denmark)

    Kueny-Stotz, Marie; Chassaing, Stefan; Brouillard, Raymond

    2008-01-01

    The synthesis of a series of derivatized flavylium cations was undertaken and the affinity to the benzodiazepine binding site of the GABA-A receptor evaluated. The observed high affinity for some derivatives (sub-muM range) was explained by an in vitro transformation of the flavylium cations into...

  13. DISC1 Protein Regulates γ-Aminobutyric Acid, Type A (GABAA) Receptor Trafficking and Inhibitory Synaptic Transmission in Cortical Neurons.

    Science.gov (United States)

    Wei, Jing; Graziane, Nicholas M; Gu, Zhenglin; Yan, Zhen

    2015-11-13

    Association studies have suggested that Disrupted-in-Schizophrenia 1 (DISC1) confers a genetic risk at the level of endophenotypes that underlies many major mental disorders. Despite the progress in understanding the significance of DISC1 at neural development, the mechanisms underlying DISC1 regulation of synaptic functions remain elusive. Because alterations in the cortical GABA system have been strongly linked to the pathophysiology of schizophrenia, one potential target of DISC1 that is critically involved in the regulation of cognition and emotion is the GABAA receptor (GABAAR). We found that cellular knockdown of DISC1 significantly reduced GABAAR-mediated synaptic and whole-cell current, whereas overexpression of wild-type DISC1, but not the C-terminal-truncated DISC1 (a schizophrenia-related mutant), significantly increased GABAAR currents in pyramidal neurons of the prefrontal cortex. These effects were accompanied by DISC1-induced changes in surface GABAAR expression. Moreover, the regulation of GABAARs by DISC1 knockdown or overexpression depends on the microtubule motor protein kinesin 1 (KIF5). Our results suggest that DISC1 exerts an important effect on GABAergic inhibitory transmission by regulating KIF5/microtubule-based GABAAR trafficking in the cortex. The knowledge gained from this study would shed light on how DISC1 and the GABA system are linked mechanistically and how their interactions are critical for maintaining a normal mental state. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

    Science.gov (United States)

    Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

    2016-06-07

    Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

  15. Influence of age, body temperature, GABAA receptor inhibition and caffeine on the Hering-Breuer inflation reflex in unanesthetized rat pups.

    Science.gov (United States)

    Arnal, Ashley V; Gore, Julie L; Rudkin, Alison; Bartlett, Donald; Leiter, J C

    2013-03-01

    We measured the duration of apnea induced by sustained end-inspiratory lung inflation (the Hering Breuer Reflex, HBR) in unanesthetized infant rat pups aged 4 days (P4) to P20 at body temperatures of 32°C and 36°C. The expiratory prolongation elicited by the HBR lasted longer in the younger pups and lasted longer at the higher body temperature. Blockade of adenosine receptors by caffeine following injection into the cisterna magna (ICM) significantly blunted the thermal prolongation of the HBR. Blockade of gama-amino-butyric acid A (GABAA) receptors by pre-treatment with ICM bicuculline had no effect on the HBR duration at either body temperature. To test the hypothesis that developmental maturation of GABAergic inhibition of breathing was modifying the response to bicuculline, we pretreated rat pups with systemically administered bumetanide to lower the intracellular chloride concentration, and repeated the bicuculline studies. Bicuculline still did not alter the HBR at either temperature after bumetanide treatment. We administered PSB-36, a selective adenosine A1 receptor antagonist, and this drug treatment did not modify the HBR. We conclude that caffeine blunts the thermal prolongation of the HBR, probably by blocking adenosine A2a receptors. The thermally sensitive adenosinergic prolongation of the HBR in these intact animals does not seem to depend on GABAA receptors. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Serum as a modulator of lipoplex-mediated gene transfection : dependence of amphiphile, cell type and complex stability

    NARCIS (Netherlands)

    Audouy, S; Molema, G; de Leij, L; Hoekstra, D

    2000-01-01

    Background Cationic liposomes belong to the family of non-viral vectors for gene delivery. Despite several drawbacks, such as low efficiency compared to viruses and inactivation by serum, cationic liposomes remain a promising tool for gene therapy. Therefore further investigation of the mechanism of

  17. The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

    Science.gov (United States)

    Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

    2013-07-01

    To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. GABA(A) receptor antagonism in the ventrocaudal periaqueductal gray increases anxiety in the anxiety-resistant postpartum rat.

    Science.gov (United States)

    Miller, Stephanie M; Piasecki, Christopher C; Peabody, Mitchell F; Lonstein, Joseph S

    2010-06-01

    Postpartum mammals show suppressed anxiety, which is necessary for their ability to appropriately care for offspring. It is parsimonious to suggest that the neurobiological basis of this reduced anxiety is similar to that of non-parturient animals, involving GABA(A) receptor activity in sites including the midbrain periaqueductal gray (PAG). In Experiment 1, postpartum and diestrous virgin female rats received an intraperitoneal injection of the GABA(A) receptor antagonist (+)-bicuculline (0, 2 and 4 mg/kg) and anxiety-related behavior was assessed with an elevated plus maze. The 4 mg/kg dose of (+)-bicuculline significantly increased anxiety-related behavior, particularly in the postpartum females. Experiment 2 revealed that bicuculline's action was within the central nervous system, because anxiety in neither dams nor virgins was significantly affected by intraperitoneal injection of bicuculline methiodide (0, 2 and 6 mg/kg), which does not readily cross the blood-brain-barrier. In Experiment 3, bicuculline methiodide (2.5 ng/side) was directly infused into the ventrocaudal PAG (cPAGv) and significantly increased dams' anxiety compared to saline-infused controls. These studies expand our knowledge of how GABA(A) receptor modulators affect anxiety behaviors in postpartum rats to the widely-used elevated plus maze, and indicate that the postpartum suppression of anxiety is in part a consequence of elevated GABAergic neurotransmission in the cPAGv. Copyright 2010 Elsevier Inc. All rights reserved.

  19. RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation

    Science.gov (United States)

    van Nierop, Pim; Vormer, Tinke L.; Foijer, Floris; Verheij, Joanne; Lodder, Johannes C.; Andersen, Jesper B.; Mansvelder, Huibert D.; te Riele, Hein

    2018-01-01

    To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo. PMID:29787571

  20. The association between etanercept serum concentration and psoriasis severity is highly age-dependent.

    Science.gov (United States)

    Detrez, Iris; Van Steen, Kristel; Segaert, Siegfried; Gils, Ann

    2017-06-01

    The association between etanercept serum concentration and psoriasis disease severity is poorly investigated, and currently etanercept serum concentration monitoring that is aiming to optimize the psoriasis treatment lacks evidence. In this prospective study, we investigated the relation between etanercept exposure and disease severity via measuring etanercept concentrations at five consecutive time points in 56 psoriasis patients. Disease severity assessments included the Psoriasis Area and Severity Index (PASI), body surface area (BSA) and Physician Global Assessment (PGA), and etanercept and anti-etanercept antibody concentrations were determined every 3 months for a period of 1 year. The present study demonstrated that the association between etanercept concentration and psoriasis severity is age-dependent: when patients were stratified into three groups, patients in the youngest age group (-50 years) showed a lower PASI at a higher etanercept concentration (β = -0.26), whereas patients in the oldest age group (+59 years) showed the opposite trend (β =0.22). Similar age effects were observed in the relation of etanercept concentration with BSA ( P =0.02) and PGA ( P =0.02). The influence of age and length of time in therapy on the etanercept concentration-disease severity relation was unaffected by body mass index (BMI) or any other possible confounder. Incidence of anti-etanercept antibodies was low (2%). The age-dependent relation between etanercept serum concentrations is both unexpected and intriguing and needs further investigation. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  1. Maladaptive perfectionism as mediator among psychological control, eating disorders, and exercise dependence symptoms in habitual exerciser.

    Science.gov (United States)

    Costa, Sebastiano; Hausenblas, Heather A; Oliva, Patrizia; Cuzzocrea, Francesca; Larcan, Rosalba

    2016-03-01

    Background and aims The current study examined the mediating role of maladaptive perfectionism among parental psychological control, eating disorder symptoms, and exercise dependence symptoms by gender in habitual exercisers. Methods Participants were 348 Italian exercisers (n = 178 men and n = 170 women; M age = 20.57, SD = 1.13) who completed self-report questionnaires assessing their parental psychological control, maladaptive perfectionism, eating disorder symptoms, and exercise dependence symptoms. Results Results of the present study confirmed the mediating role of maladaptive perfectionism for eating disorder and exercise dependence symptoms for the male and female exercisers in the maternal data. In the paternal data, maladaptive perfectionism mediated the relationships between paternal psychological control and eating disorder and exercise dependence symptoms as full mediator for female participants and as partial mediator for male participants. Discussion Findings of the present study suggest that it may be beneficial to consider dimensions of maladaptive perfectionism and parental psychological control when studying eating disorder and exercise dependence symptoms in habitual exerciser.

  2. Structural changes at the myrtenol backbone reverse its positive allosteric potential into inhibitory GABAA receptor modulation

    DEFF Research Database (Denmark)

    Milanos, Sinem; Kuenzel, Katharina; Gilbert, Daniel F

    2017-01-01

    monoterpenes, e.g. myrtenol as positive allosteric modulator at α1β2 GABAA receptors. Here, along with pharmacophore-based virtual screening studies, we demonstrate that scaffold modifications of myrtenol resulted in loss of modulatory activity. Two independent approaches, fluorescence-based compound analysis...

  3. Effect of hydromorphone hydrochloride combined with bupivacaine combined spinal-epidural anesthesia on serum pain mediators and placental hypoxia molecules after cesarean section

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hui Yuan; Jian-Guo Xia; Xiao-Yang Jiang

    2017-01-01

    Objective:To study the effect of hydromorphone hydrochloride combined with bupivacaine combined spinal-epidural anesthesia on serum pain mediators and placental hypoxia molecules after cesarean section.Methods: 126 women accepting cesarean section in our hospital between May 2013 and December 2015 were selected as the research subjects and randomly divided into two groups, observation group of subjects received hydromorphone hydrochloride combined with bupivacaine combined spinal-epidural anesthesia and control group of subjects accepted bupivacaine combined spinal-epidural anesthesia. 1, 3 and 5 d after delivery, serum was collected to determine the levels of pain mediators; the placenta tissue was collected to detect the levels of oxidative stress molecules and endoplasmic reticulum stress molecules.Results: 1, 3 and 5 d after delivery, serum substance P (SP),β-endorphin (β-EP), 5-hydroxytryptamine (5-HT), nitric oxide (NO) and norepinephrine (NE) levels of observation group were significantly lower than those of control group whileβ-EP levels were significantly higher than those of control group (P<0.05); reactive oxide species (ROS), reactive nitrogen species (RNS), GRP78, ERdj1, CHOP and ASK1 levels in placenta tissue of observation group were significantly lower than those of control group while glutathione peroxidase (GPx), catalase (CAT) and vitamin C (VitC) levels were significantly higher than those of control group (P<0.05).Conclusions: Hydromorphone hydrochloride combined with bupivacaine combined spinal-epidural anesthesia can adjust the pain mediator secretion, relieve postoperative pain and inhibit oxidative stress and endoplasmic reticulum stress.

  4. Olfactory bulb glomerular NMDA receptors mediate olfactory nerve potentiation and odor preference learning in the neonate rat.

    Directory of Open Access Journals (Sweden)

    Rebecca Lethbridge

    Full Text Available Rat pup odor preference learning follows pairing of bulbar beta-adrenoceptor activation with olfactory input. We hypothesize that NMDA receptor (NMDAR-mediated olfactory input to mitral cells is enhanced during training, such that increased calcium facilitates and shapes the critical cAMP pattern. Here, we demonstrate, in vitro, that olfactory nerve stimulation, at sniffing frequencies, paired with beta-adrenoceptor activation, potentiates olfactory nerve-evoked mitral cell firing. This potentiation is blocked by a NMDAR antagonist and by increased inhibition. Glomerular disinhibition also induces NMDAR-sensitive potentiation. In vivo, in parallel, behavioral learning is prevented by glomerular infusion of an NMDAR antagonist or a GABA(A receptor agonist. A glomerular GABA(A receptor antagonist paired with odor can induce NMDAR-dependent learning. The NMDA GluN1 subunit is phosphorylated in odor-specific glomeruli within 5 min of training suggesting early activation, and enhanced calcium entry, during acquisition. The GluN1 subunit is down-regulated 3 h after learning; and at 24 h post-training the GluN2B subunit is down-regulated. These events may assist memory stability. Ex vivo experiments using bulbs from trained rat pups reveal an increase in the AMPA/NMDA EPSC ratio post-training, consistent with an increase in AMPA receptor insertion and/or the decrease in NMDAR subunits. These results support a model of a cAMP/NMDA interaction in generating rat pup odor preference learning.

  5. Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAA Receptors with Different γ Subunits

    Directory of Open Access Journals (Sweden)

    Zhiwen Ye

    2017-04-01

    Full Text Available Cell-type specific differences in the kinetics of inhibitory postsynaptic conductance changes (IPSCs are believed to impact upon network dynamics throughout the brain. Much attention has focused on how GABAA receptor (GABAAR α and β subunit diversity will influence IPSC kinetics, but less is known about the influence of the γ subunit. We have examined whether GABAAR γ subunit heterogeneity influences IPSC properties in the thalamus. The γ2 subunit gene was deleted from GABAARs selectively in the dorsal lateral geniculate nucleus (dLGN. The removal of the γ2 subunit from the dLGN reduced the overall spontaneous IPSC (sIPSC frequency across all relay cells and produced an absence of IPSCs in a subset of relay neurons. The remaining slower IPSCs were both insensitive to diazepam and zinc indicating the absence of the γ2 subunit. Because these slower IPSCs were potentiated by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, we propose these IPSCs involve γ1 subunit-containing GABAAR activation. Therefore, γ subunit heterogeneity appears to influence the kinetics of GABAAR-mediated synaptic transmission in the visual thalamus in a cell-selective manner. We suggest that activation of γ1 subunit-containing GABAARs give rise to slower IPSCs in general, while faster IPSCs tend to be mediated by γ2 subunit-containing GABAARs.

  6. Early continuous white noise exposure alters auditory spatial sensitivity and expression of GAD65 and GABAA receptor subunits in rat auditory cortex.

    Science.gov (United States)

    Xu, Jinghong; Yu, Liping; Cai, Rui; Zhang, Jiping; Sun, Xinde

    2010-04-01

    Sensory experiences have important roles in the functional development of the mammalian auditory cortex. Here, we show how early continuous noise rearing influences spatial sensitivity in the rat primary auditory cortex (A1) and its underlying mechanisms. By rearing infant rat pups under conditions of continuous, moderate level white noise, we found that noise rearing markedly attenuated the spatial sensitivity of A1 neurons. Compared with rats reared under normal conditions, spike counts of A1 neurons were more poorly modulated by changes in stimulus location, and their preferred locations were distributed over a larger area. We further show that early continuous noise rearing induced significant decreases in glutamic acid decarboxylase 65 and gamma-aminobutyric acid (GABA)(A) receptor alpha1 subunit expression, and an increase in GABA(A) receptor alpha3 expression, which indicates a returned to the juvenile form of GABA(A) receptor, with no effect on the expression of N-methyl-D-aspartate receptors. These observations indicate that noise rearing has powerful adverse effects on the maturation of cortical GABAergic inhibition, which might be responsible for the reduced spatial sensitivity.

  7. Gaba mediated long-term depression (LTD) in the rat medial vestibular nuclei.

    Science.gov (United States)

    Grassi, S; Della Torre, G; Zampolini, M; Pettorossi, V E

    1995-01-01

    As previously demonstrated, high frequency stimulation (HFS) of the primary vestibular afferents always induces a clear, long lasting depression of the polysynaptic (N2) component of the field potentials recorded in the dorsal portion of the medial vestibular nuclei (MVN). The induction of the HFS effect was mediated by the activation of glutamate NMDA receptors, since it was blocked by AP5. The mechanisms at the basis of such a depression were studied. Our results demonstrate that Gaba, acting on both GabaA and GabaB receptors, is involved in mediating this phenomenon. In fact, HFS applied during Bicuculline and Saclofen perfusion, was no longer able to induce an N2 depression, but provoked a slight potentiation. However, the N2 depression clearly emerged after drug wash-out. Furthermore, Bicuculline and Saclofen fully abolished the N2 depression and highlighted the potentiation, when administered after HFS. The possibility that the N2 depression is the result of a homosynaptic LTD can be excluded on the basis of our results. On the contrary, our findings suggest that the depression is due to an enhancement of the Gaba inhibitory effect due to an HFS dependent increase in gabaergic interneuron activity, which resets vestibular neuron excitability at a lower level.

  8. The role of exercise dependence for the relationship between exercise behavior and eating pathology: mediator or moderator?

    Science.gov (United States)

    Cook, Brian J; Hausenblas, Heather A

    2008-05-01

    Our study examined the potential mediating or moderating effect of exercise dependence on the exercise-eating pathology relationship. Female university students (N = 330) completed Internet-based self-report measures of exercise behavior, exercise dependence, and eating pathology. Exercise dependence served as a mediator for the relationship between exercise and eating pathology. This unidirectional causal model suggests that an individual's pathological motivation or compulsion to exercise is the critical mediating component in the exercise-eating pathology relationship. The best target for removing the link between exercise behavior and eating pathology may be reformulating exercise dependence symptoms.

  9. Strain- and Substrate-Dependent Redox Mediator and Electricity Production by Pseudomonas aeruginosa.

    Science.gov (United States)

    Bosire, Erick M; Blank, Lars M; Rosenbaum, Miriam A

    2016-08-15

    Pseudomonas aeruginosa is an important, thriving member of microbial communities of microbial bioelectrochemical systems (BES) through the production of versatile phenazine redox mediators. Pure culture experiments with a model strain revealed synergistic interactions of P. aeruginosa with fermenting microorganisms whereby the synergism was mediated through the shared fermentation product 2,3-butanediol. Our work here shows that the behavior and efficiency of P. aeruginosa in mediated current production is strongly dependent on the strain of P. aeruginosa We compared levels of phenazine production by the previously investigated model strain P. aeruginosa PA14, the alternative model strain P. aeruginosa PAO1, and the BES isolate Pseudomonas sp. strain KRP1 with glucose and the fermentation products 2,3-butanediol and ethanol as carbon substrates. We found significant differences in substrate-dependent phenazine production and resulting anodic current generation for the three strains, with the BES isolate KRP1 being overall the best current producer and showing the highest electrochemical activity with glucose as a substrate (19 μA cm(-2) with ∼150 μg ml(-1) phenazine carboxylic acid as a redox mediator). Surprisingly, P. aeruginosa PAO1 showed very low phenazine production and electrochemical activity under all tested conditions. Microbial fuel cells and other microbial bioelectrochemical systems hold great promise for environmental technologies such as wastewater treatment and bioremediation. While there is much emphasis on the development of materials and devices to realize such systems, the investigation and a deeper understanding of the underlying microbiology and ecology are lagging behind. Physiological investigations focus on microorganisms exhibiting direct electron transfer in pure culture systems. Meanwhile, mediated electron transfer with natural redox compounds produced by, for example, Pseudomonas aeruginosa might enable an entire microbial

  10. Context-dependent memory following recurrent hypoglycaemia in non-diabetic rats is mediated via glucocorticoid signalling in the dorsal hippocampus.

    Science.gov (United States)

    Osborne, Danielle M; O'Leary, Kelsey E; Fitzgerald, Dennis P; George, Alvin J; Vidal, Michael M; Anderson, Brian M; McNay, Ewan C

    2017-01-01

    Recurrent hypoglycaemia is primarily caused by repeated over-administration of insulin to patients with diabetes. Although cognition is impaired during hypoglycaemia, restoration of euglycaemia after recurrent hypoglycaemia is associated with improved hippocampally mediated memory. Recurrent hypoglycaemia alters glucocorticoid secretion in response to hypoglycaemia; glucocorticoids are well established to regulate hippocampal processes, suggesting a possible mechanism for recurrent hypoglycaemia modulation of subsequent cognition. We tested the hypothesis that glucocorticoids within the dorsal hippocampus might mediate the impact of recurrent hypoglycaemia on hippocampal cognitive processes. We characterised changes in the dorsal hippocampus at several time points to identify specific mechanisms affected by recurrent hypoglycaemia, using a well-validated 3 day model of recurrent hypoglycaemia either alone or with intrahippocampal delivery of glucocorticoid (mifepristone) and mineralocorticoid (spironolactone) receptor antagonists prior to each hypoglycaemic episode. Recurrent hypoglycaemia enhanced learning and also increased hippocampal expression of glucocorticoid receptors, serum/glucocorticoid-regulated kinase 1, cyclic AMP response element binding (CREB) phosphorylation, and plasma membrane levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptors. Both hippocampus-dependent memory enhancement and the molecular changes were reversed by glucocorticoid receptor antagonist treatment. These results indicate that increased glucocorticoid signalling during recurrent hypoglycaemia produces several changes in the dorsal hippocampus that are conducive to enhanced hippocampus-dependent contextual learning. These changes appear to be adaptive, and in addition to supporting cognition may reduce damage otherwise caused by repeated exposure to severe hypoglycaemia.

  11. Structural basis for ligand recognition at the benzodiazepine binding site of GABAA alpha 3 receptor, and pharmacophore-based virtual screening approach.

    Science.gov (United States)

    Vijayan, R S K; Ghoshal, Nanda

    2008-10-01

    Given the heterogeneity of GABA(A) receptor, the pharmacological significance of identifying subtype selective modulators is increasingly being recognized. Thus, drugs selective for GABA(A) alpha(3) receptors are expected to display fewer side effects than the drugs presently in clinical use. Hence we carried out 3D QSAR (three-dimensional quantitative structure-activity relationship) studies on a series of novel GABA(A) alpha(3) subtype selective modulators to gain more insight into subtype affinity. To identify the 3D functional attributes required for subtype selectivity, a chemical feature-based pharmacophore, primarily based on selective ligands representing diverse structural classes was generated. The obtained pseudo receptor model of the benzodiazepine binding site revealed a binding mode akin to "Message-Address" concept. Scaffold hopping was carried out across multi-conformational May Bridge database for the identification of novel chemotypes. Further a focused data reduction approach was employed to choose a subset of enriched compounds based on "Drug likeness" and "Similarity-based" methods. These results taken together could provide impetus for rational design and optimization of more selective and high affinity leads with a potential to have decreased adverse effects.

  12. BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella

    Science.gov (United States)

    Andree, Maria; Seeger, Jens M; Schüll, Stephan; Coutelle, Oliver; Wagner-Stippich, Diana; Wiegmann, Katja; Wunderlich, Claudia M; Brinkmann, Kerstin; Broxtermann, Pia; Witt, Axel; Fritsch, Melanie; Martinelli, Paola; Bielig, Harald; Lamkemeyer, Tobias; Rugarli, Elena I; Kaufmann, Thomas; Sterner-Kock, Anja; Wunderlich, F Thomas; Villunger, Andreas; Martins, L Miguel; Krönke, Martin; Kufer, Thomas A; Utermöhlen, Olaf; Kashkar, Hamid

    2014-01-01

    The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization. PMID:25056906

  13. Effect of hydromorphone hydrochloride combined with bupivacaine combined spinal-epidural anesthesia on serum pain mediators and placental hypoxia molecules after cesarean section

    Directory of Open Access Journals (Sweden)

    Xiao-Hui Yuan

    2017-05-01

    Full Text Available Objective: To study the effect of hydromorphone hydrochloride combined with bupivacaine combined spinal-epidural anesthesia on serum pain mediators and placental hypoxia molecules after cesarean section. Methods: 126 women accepting cesarean section in our hospital between May 2013 and December 2015 were selected as the research subjects and randomly divided into two groups, observation group of subjects received hydromorphone hydrochloride combined with bupivacaine combined spinal-epidural anesthesia and control group of subjects accepted bupivacaine combined spinal-epidural anesthesia. 1, 3 and 5 d after delivery, serum was collected to determine the levels of pain mediators; the placenta tissue was collected to detect the levels of oxidative stress molecules and endoplasmic reticulum stress molecules. Results: 1, 3 and 5 d after delivery, serum substance P (SP, β-endorphin (β-EP, 5-hydroxytryptamine (5-HT, nitric oxide (NO and norepinephrine (NE levels of observation group were significantly lower than those of control group while β-EP levels were significantly higher than those of control group (P<0.05; reactive oxide species (ROS, reactive nitrogen species (RNS, GRP78, ERdj1, CHOP and ASK1 levels in placenta tissue of observation group were significantly lower than those of control group while glutathione peroxidase (GPx, catalase (CAT and vitamin C (VitC levels were significantly higher than those of control group (P<0.05. Conclusions: Hydromorphone hydrochloride combined with bupivacaine combined spinal-epidural anesthesia can adjust the pain mediator secretion, relieve postoperative pain and inhibit oxidative stress and endoplasmic reticulum stress.

  14. The insulinogenic effect of whey protein is partially mediated by a direct effect of amino acids and GIP on β-cells

    DEFF Research Database (Denmark)

    Salehi, Albert; Gunnerud, Ulrika; Muhammed, Sarheed J

    2012-01-01

    Whey protein increases postprandial serum insulin levels. This has been associated with increased serum levels of leucine, isoleucine, valine, lysine, threonine and the incretin hormone glucose-dependent insulinotropic polypeptide (GIP). We have examined the effects of these putative mediators...... of whey's action on insulin secretion from isolated mouse Langerhans islets....

  15. Advances in therapeutic Fc engineering - modulation of IgG associated effector functions and serum half-life

    Directory of Open Access Journals (Sweden)

    Abhishek Saxena

    2016-12-01

    Full Text Available Today monoclonal immunoglobulin gamma (IgG antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs are achieved through both its antigen binding fragment (Fab and crystallizable fragment (Fc. Fab can specifically recognize tumor associated antigen (TAA and thus modulate TAA-linked downstream signaling pathways that may lead to inhibition of tumor growth, induction of tumor apoptosis and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity (ADCC, complement-dependent cytotoxicity (CDC and antibody dependent cell-mediated phagocytosis (ADCP. Moreover, Fc is the region interacting with the neonatal Fc receptor (FcRn in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anti-cancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering based mAbs under clinical trials.

  16. The assessment of serum-mediated phagocytosis of necrotic material by polymorphonuclear leukocytes to diagnose and predict the clinical features of systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Compagno, Michele; Gullstrand, Birgitta; Jacobsen, Søren

    2016-01-01

    BACKGROUND: Serum-mediated phagocytosis of antibody- and complement-opsonized necrotic cell material (NCM) by polymorphonuclear leukocytes can be quantified by using a flow cytometry-based assay. The phagocytosis of necrotic cell material (PNC) assay parallels the well-known lupus erythematosus c...

  17. GABA(A) receptor modulation during adolescence alters adult ethanol intake and preference in rats.

    Science.gov (United States)

    Hulin, Mary W; Amato, Russell J; Winsauer, Peter J

    2012-02-01

    To address the hypothesis that GABA(A) receptor modulation during adolescence may alter the abuse liability of ethanol during adulthood, the effects of adolescent administration of both a positive and negative GABA(A) receptor modulator on adult alcohol intake and preference were assessed. Three groups of adolescent male rats received 12 injections of lorazepam (3.2 mg/kg), dehydroepiandrosterone (DHEA, 56 mg/kg), or vehicle on alternate days starting on postnatal day (PD) 35. After this time, the doses were increased to 5.6 and 100 mg/kg, respectively, for 3 more injections on alternate days. Subjects had access to 25 to 30 g of food daily, during the period of the first 6 injections, and 18 to 20 g thereafter. Food intake of each group was measured 60 minutes after food presentation, which occurred immediately after drug administration on injection days or at the same time of day on noninjection days. When subjects reached adulthood (PD 88), ethanol preference was determined on 2 separate occasions, an initial 3-day period and a 12-day period, in which increasing concentrations of ethanol were presented. During each preference test, intake of water, saccharin, and an ethanol/saccharin solution was measured after each 23-hour access period. During adolescence, lorazepam increased 60-minute food intake, and this effect was enhanced under the more restrictive feeding schedule. DHEA had the opposite effect on injection days, decreasing food intake compared with noninjection days. In adulthood, the lorazepam-treated group preferred the 2 lowest concentrations of ethanol/saccharin more than saccharin alone compared with vehicle-treated subjects, which showed no preference for any concentration of ethanol/saccharin over saccharin. DHEA-treated subjects showed no preference among the 3 solutions. These data demonstrate that GABA(A) receptor modulation during adolescence can alter intake and preference for ethanol in adulthood and highlights the importance of drug history

  18. Exercise dependence as a mediator of the exercise and eating disorders relationship: a pilot study.

    Science.gov (United States)

    Cook, Brian; Hausenblas, Heather; Crosby, Ross D; Cao, Li; Wonderlich, Stephen A

    2015-01-01

    Excessive exercise is a common feature of eating disorders (ED) and is associated with earlier ED onset, more ED symptoms, and higher persistence of ED behavior. Research indicates that exercise amount alone is not associated with ED. The purpose of this study was to investigate pathological attitudes and behaviors related to exercise (e.g., exercise dependence) as a mediator of the exercise and ED relationship. Participants were 43 women with an ED who completed measures of ED symptoms, exercise behavior, and exercise dependence. Analyses were conducted using the indirect bootstrapping method for examining mediation. Exercise dependence mediated the relationship between exercise and ED. This mediation model accounted for 14.34% of the variance in the relationship. Our results extend the literature by offering preliminary evidence of a psychological variable that may be a candidate for future interventions on the exercise and ED relationship. Implications and suggestions for future research are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Relationship between cobalamin-dependent metabolites and both serum albumin and alpha1 -proteinase inhibitor concentrations in hypocobalaminemic dogs of 7 different breeds.

    Science.gov (United States)

    Grützner, Niels; Suchodolski, Jan S; Steiner, Jörg M

    2014-12-01

    Increased serum concentrations of homocysteine (HCY) and methylmalonic acid (MMA), the 2 main cobalamin-dependent metabolites, as well as decreased serum albumin and canine alpha1 -proteinase inhibitor (cα1 -PI) concentrations have previously been described in hypocobalaminemic dogs with gastrointestinal disease. However, no studies have been conducted to evaluate potential relationships between these serum biomarkers. The aim of this study was to evaluate the relationship between HCY and MMA, 2 cobalamin-dependent metabolites, and both serum albumin and cα1 -PI concentrations in hypocobalaminemic dogs. Serum samples from 285 dogs including 7 different breeds (Beagle, Boxer, Cocker Spaniel, German Shepherd, Labrador Retriever, Chinese Shar-Pei, and Yorkshire Terrier) with hypocobalaminemia were used. Serum HCY, MMA, albumin, and cα1 -PI concentrations were determined. There was a significant correlation between serum HCY and albumin concentrations, as well as serum HCY and cα1 -PI concentrations (ρ = 0.62 and ρ = 0.37, respectively; P  .05). In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, and serum HCY and MMA concentrations in Chinese Shar-Peis with hypocobalaminemia. This study shows a correlation between serum albumin and cα1 -PI and HCY concentrations, but not with serum MMA concentration in dogs with hypocobalaminemia. In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, as well as serum HCY and MMA concentrations in Chinese Shar-Peis, emphasizing the unique metabolic interactions in those dog breeds affected by hypocobalaminemia. © 2014 American Society for Veterinary Clinical Pathology.

  20. Mediator-dependent Nuclear Receptor Functions

    Science.gov (United States)

    Chen, Wei; Roeder, Robert

    2011-01-01

    As gene-specific transcription factors, nuclear hormone receptors are broadly involved in many important biological processes. Their function on target genes requires the stepwise assembly of different coactivator complexes that facilitate chromatin remodeling and subsequent preinitiation complex (PIC) formation and function. Mediator has proved to be a crucial, and general, nuclear receptor-interacting coactivator, with demonstrated functions in transcription steps ranging from chromatin remodeling to subsequent PIC formation and function. Here we discuss (i) our current understanding of pathways that nuclear receptors and other interacting cofactors employ to recruit Mediator to target gene enhancers and promoters, including conditional requirements for the strong NR-Mediator interactions mediated by the NR AF2 domain and the MED1 LXXLLL motifs and (ii) mechanisms by which Mediator acts to transmit signals from enhancer-bound nuclear receptors to the general transcription machinery at core promoters to effect PIC formation and function. PMID:21854863

  1. Paradoxical effects of GABA-A modulators may explain sex steroid induced negative mood symptoms in some persons

    NARCIS (Netherlands)

    Bäckström, T.; Haage, D.; Löfgren, M.; Johansson, I. M.; Strömberg, J.; Nyberg, S.; Andréen, L.; Ossewaarde, L.; van Wingen, G. A.; Turkmen, S.; Bengtsson, S. K.

    2011-01-01

    Some women have negative mood symptoms, caused by progestagens in hormonal contraceptives or sequential hormone therapy or by progesterone in the luteal phase of the menstrual cycle, which may be attributed to metabolites acting on the GABA-A receptor. The GABA system is the major inhibitory system

  2. Nootropic agents enhance the recruitment of fast GABAA inhibition in rat neocortex.

    Science.gov (United States)

    Ling, Douglas S F; Benardo, Larry S

    2005-07-01

    It is widely believed that nootropic (cognition-enhancing) agents produce their therapeutic effects by augmenting excitatory synaptic transmission in cortical circuits, primarily through positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors (AMPARs). However, GABA-mediated inhibition is also critical for cognition, and enhanced GABA function may be likewise therapeutic for cognitive disorders. Could nootropics act through such a mechanism as well? To address this question, we examined the effects of nootropic agents on excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) recorded from layer V pyramidal cells in acute slices of somatosensory cortex. Aniracetam, a positive modulator of AMPA/kainate receptors, increased the peak amplitude of evoked EPSCs and the amplitude and duration of polysynaptic fast IPSCs, manifested as a greater total charge carried by IPSCs. As a result, the EPSC/IPSC ratio of total charge was decreased, representing a shift in the excitation-inhibition balance that favors inhibition. Aniracetam did not affect the magnitude of either monosynaptic IPSCs (mono-IPSCs) recorded in the presence of excitatory amino acid receptor antagonists, or miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin. However, the duration of both mono-IPSCs and mIPSCs was prolonged, suggesting that aniracetam also directly modulates GABAergic transmission. Cyclothiazide, a preferential modulator of AMPAR function, enhanced the magnitude and duration of polysynaptic IPSCs, similar to aniracetam, but did not affect mono-IPSCs. Concanavalin A, a kainate receptor modulator, had little effect on EPSCs or IPSCs, suggesting there was no contribution from kainate receptor activity. These findings indicate that AMPAR modulators strengthen inhibition in neocortical pyramidal cells, most likely by altering the kinetics of AMPARs on synaptically connected interneurons and possibly by modulating GABA(A) receptor responses

  3. Elevated baseline serum glutamate as a pharmacometabolomic biomarker for acamprosate treatment outcome in alcohol-dependent subjects

    Science.gov (United States)

    Nam, H W; Karpyak, V M; Hinton, D J; Geske, J R; Ho, A M C; Prieto, M L; Biernacka, J M; Frye, M A; Weinshilboum, R M; Choi, D-S

    2015-01-01

    Acamprosate has been widely used since the Food and Drug Administration approved the medication for treatment of alcohol use disorders (AUDs) in 2004. Although the detailed molecular mechanism of acamprosate remains unclear, it has been largely known that acamprosate inhibits glutamate action in the brain. However, AUD is a complex and heterogeneous disorder. Thus, biomarkers are required to prescribe this medication to patients who will have the highest likelihood of responding positively. To identify pharmacometabolomic biomarkers of acamprosate response, we utilized serum samples from 120 alcohol-dependent subjects, including 71 responders (maintained continuous abstinence) and 49 non-responders (any alcohol use) during 12 weeks of acamprosate treatment. Notably, baseline serum glutamate levels were significantly higher in responders compared with non-responders. Importantly, serum glutamate levels of responders are normalized after acamprosate treatment, whereas there was no significant glutamate change in non-responders. Subsequent functional studies in animal models revealed that, in the absence of alcohol, acamprosate activates glutamine synthetase, which synthesizes glutamine from glutamate and ammonia. These results suggest that acamprosate reduces serum glutamate levels for those who have elevated baseline serum glutamate levels among responders. Taken together, our findings demonstrate that elevated baseline serum glutamate levels are a potential biomarker associated with positive acamprosate response, which is an important step towards development of a personalized approach to treatment for AUD. PMID:26285131

  4. Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors

    Science.gov (United States)

    Kletke, Olaf; Gisselmann, Guenter; May, Andrea; Hatt, Hanns; A. Sergeeva, Olga

    2013-01-01

    Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α1/2β1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant αxβ1δ-GABAAR, we generated a chimeric γ2 subunit carrying the δ subunit motif around F77 (MTVFLH). At α1/2β1γ2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β3-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABAA receptors. Our study sheds new light on the β1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions. PMID:23637894

  5. Acute sleep deprivation preconditions the heart against ischemia/ reperfusion injury: the role of central GABA-A receptors.

    Science.gov (United States)

    Parsa, Hoda; Imani, Alireza; Faghihi, Mahdieh; Riahi, Esmail; Badavi, Mohammad; Shakoori, Abbas; Rastegar, Tayebeh; Aghajani, Marjan; Rajani, Sulail Fatima

    2017-11-01

    Central γ-aminobutyric acid (GABA) neurotransmission modulates cardiovascular functions and sleep. Acute sleep deprivation (ASD) affects functions of various body organs via different mechanisms. Here, we evaluated the effect of ASD on cardiac ischemia/reperfusion injury (IRI), and studied the role of GABA-A receptor inhibition in central nucleus of amygdala (CeA) by assessing nitric oxide (NO) and oxidative stress. The CeA in sixty male Wistar rats was cannulated for saline or bicuculline (GABA-A receptor antagonist) administration. All animals underwent 30 min of coronary occlusion (ischemia), followed by 2 hr reperfusion (IR). The five experimental groups (n=12) included are as follows: IR: received saline; BIC+IR: received Bicuculline; MLP+IR: received saline, followed by the placement of animals in an aquarium with multiple large platforms; ASD+IR: underwent ASD in an aquarium with multiple small platforms; and BIC+ASD+IR: received bicuculline prior to ASD. Bicuculline administration increased the malondialdehyde levels and infarct size, and decreased the NO metabolites levels and endothelial nitric oxide synthase (eNOS) gene expression in infarcted and non-infarcted areas in comparison to IR group. ASD reduced malondialdehyde levels and infarct size and increased NO metabolites, corticosterone levels and eNOS expression in infarcted and non-infarcted areas as compared to the IR group. Levels of malondialdehyde were increased while levels of NO metabolites, corticosterone and eNOS expression in infarcted and non-infarcted areas were reduced in the BIC+ASD+IR as compared to the ASD+IR group. Blockade of GABA-A receptors in the CeA abolishes ASD-induced cardioprotection by suppressing oxidative stress and NO production.

  6. Acute sleep deprivation preconditions the heart against ischemia/ reperfusion injury: the role of central GABA-A receptors

    Directory of Open Access Journals (Sweden)

    Hoda Parsa

    2017-11-01

    Full Text Available Objective(s: Central γ-aminobutyric acid (GABA neurotransmission modulates cardiovascular functions and sleep. Acute sleep deprivation (ASD affects functions of various body organs via different mechanisms. Here, we evaluated the effect of ASD on cardiac ischemia/reperfusion injury (IRI, and studied the role of GABA-A receptor inhibition in central nucleus of amygdala (CeA by assessing nitric oxide (NO and oxidative stress. Materials and Methods: The CeA in sixty male Wistar rats was cannulated for saline or bicuculline (GABA-A receptor antagonist administration. All animals underwent 30 min of coronary occlusion (ischemia, followed by 2 hr reperfusion (IR. The five experimental groups (n=12 included are as follows: IR: received saline; BIC+IR: received Bicuculline; MLP+IR: received saline, followed by the placement of animals in an aquarium with multiple large platforms; ASD+IR: underwent ASD in an aquarium with multiple small platforms; and BIC+ASD+IR: received bicuculline prior to ASD. Results: Bicuculline administration increased the malondialdehyde levels and infarct size, and decreased the NO metabolites levels and endothelial nitric oxide synthase (eNOS gene expression in infarcted and non-infarcted areas in comparison to IR group. ASD reduced malondialdehyde levels and infarct size and increased NO metabolites, corticosterone levels and eNOS expression in infarcted and non-infarcted areas as compared to the IR group. Levels of malondialdehyde were increased while levels of NO metabolites, corticosterone and eNOS expression in infarcted and non-infarcted areas were reduced in the BIC+ASD+IR as compared to the ASD+IR group. Conclusion: Blockade of GABA-A receptors in the CeA abolishes ASD-induced cardioprotection by suppressing oxidative stress and NO production.

  7. Anion transport and GABA signaling

    Directory of Open Access Journals (Sweden)

    Christian Andreas Huebner

    2013-10-01

    Full Text Available Whereas activation of GABAA receptors by GABA usually results in a hyperpolarizing influx of chloride into the neuron, the reversed chloride driving force in the immature nervous system results in a depolarizing efflux of chloride. This GABAergic depolarization is deemed to be important for the maturation of the neuronal network. The concept of a developmental GABA switch has mainly been derived from in vitro experiments and reliable in vivo evidence is still missing. As GABAA receptors are permeable for both chloride and bicarbonate, the net effect of GABA also critically depends on the distribution of bicarbonate. Whereas chloride can either mediate depolarizing or hyperpolarizing currents, bicarbonate invariably mediates a depolarizing current under physiological conditions. Intracellular bicarbonate is quickly replenished by cytosolic carbonic anhydrases. Intracellular bicarbonate levels also depend on different bicarbonate transporters expressed by neurons. The expression of these proteins is not only developmentally regulated but also differs between cell types and even subcellular regions. In this review we will summarize current knowledge about the role of some of these transporters for brain development and brain function.

  8. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Hösli, E; Belhage, B

    1991-01-01

    . At the light microscope level specific staining of GABAA-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling...

  9. Human prealbumin fraction: effects on cell-mediated immunity and tumor rejection

    International Nuclear Information System (INIS)

    Leung, K.H.; Ehrke, M.J.; Bercsenyi, K.; Mihich, E.

    1982-01-01

    The effect of human prealbumin fraction as allogeneic cell-mediated immunity in primary sensitization cultures of murine spleen cells was studied by 3H-thymidine uptake and specific 51Cr release assays. Prealbumin caused a dose-dependent augmentation of these responses. Human serum albumin, bovine serum albumin, and calf-thymosin fraction 5 had little effect. Prealbumin was active when added on day 0 or 1 but not thereafter. Prealbumin added to effector cells from immunized mice did not change their lytic activity. Prealbumin, but not human serum albumin or thymosin fraction 5, augmented secondary cell-mediated immunity in culture after primary immunization in mice. A slow growing mammary tumor line, which originated as a spontaneous mammary tumor in a DBA/2 HaDD breeder mouse, initially grows in 100% of DBA/2J mice but is then rejected in 10 to 20% of them. When prealbumin (59 microgram/day) was given subcutaneously for 2 weeks to DBA/2J mice and the tumor implanted 2 weeks later. 78% of the mice rejected the tumor and were then resistant to a rechallenge

  10. Activity-dependent changes in excitability of perirhinal cortex networks in vitro.

    Science.gov (United States)

    Biagini, Giuseppe; D'Antuono, Margherita; Inaba, Yuji; Kano, Toshiyuki; Ragsdale, David; Avoli, Massimo

    2015-04-01

    Rat brain slices comprising the perirhinal cortex (PC) and a portion of the lateral nucleus of the amygdala (LA), in standard medium, can generate synchronous oscillatory activity that is associated with action potential discharge and reflects the activation of glutamatergic and GABAergic receptors. We report here that similar synchronous oscillatory events are recorded in the PC in response to single-shock, electrical stimuli delivered in LA. In addition, we found that the latency of these responses progressively increased when the stimulus interval was varied from 10 to 1 s; for example, the response latency during stimuli delivered at 1 Hz was more than twofold longer than that seen during stimulation at 0.1 Hz. This prolongation in latency occurred after approximately 5 stimuli, attained a steady value after 24-35 stimuli, and recovered to control values 30 s after stimulation arrest. These frequency-dependent changes in latency continued to occur during NMDA receptor antagonism but weakened following application of GABAA and/or GABAB receptor blockers. Our findings identify a new type of short-term plasticity that is mediated by GABA receptor function and may play a role in decreasing neuronal network synchronization during repeated activation. We propose that this frequency-dependent adaptive mechanism influences the excitability of limbic networks, thus potentially controlling epileptiform synchronization.

  11. Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

    Science.gov (United States)

    Vela, J; Vitorica, J; Ruano, D

    2001-12-01

    We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

  12. The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

    DEFF Research Database (Denmark)

    Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

    2006-01-01

    CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

  13. Serum testosterone concentration in chloroquine- treated rats ...

    African Journals Online (AJOL)

    ONOS

    2010-07-05

    Jul 5, 2010 ... The effects of ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) were studied on serum testosterone ... chloroquine are probably mediated via the generation of free radicals. ... Effects of ascorbic acid and alpha-tocopherol on serum testosterone concentration in chloroquine-treated rats. Groups.

  14. Correction of Diabetic Hyperglycemia and Amelioration of Metabolic Anomalies by Minicircle DNA Mediated Glucose-Dependent Hepatic Insulin Production.

    Directory of Open Access Journals (Sweden)

    Tausif Alam

    Full Text Available Type 1 diabetes mellitus (T1DM is caused by immune destruction of insulin-producing pancreatic β-cells. Commonly used insulin injection therapy does not provide a dynamic blood glucose control to prevent long-term systemic T1DM-associated damages. Donor shortage and the limited long-term success of islet transplants have stimulated the development of novel therapies for T1DM. Gene therapy-based glucose-regulated hepatic insulin production is a promising strategy to treat T1DM. We have developed gene constructs which cause glucose-concentration-dependent human insulin production in liver cells. A novel set of human insulin expression constructs containing a combination of elements to improve gene transcription, mRNA processing, and translation efficiency were generated as minicircle DNA preparations that lack bacterial and viral DNA. Hepatocytes transduced with the new constructs, ex vivo, produced large amounts of glucose-inducible human insulin. In vivo, insulin minicircle DNA (TA1m treated streptozotocin (STZ-diabetic rats demonstrated euglycemia when fasted or fed, ad libitum. Weight loss due to uncontrolled hyperglycemia was reversed in insulin gene treated diabetic rats to normal rate of weight gain, lasting ∼1 month. Intraperitoneal glucose tolerance test (IPGT demonstrated in vivo glucose-responsive changes in insulin levels to correct hyperglycemia within 45 minutes. A single TA1m treatment raised serum albumin levels in diabetic rats to normal and significantly reduced hypertriglyceridemia and hypercholesterolemia. Elevated serum levels of aspartate transaminase, alanine aminotransferase, and alkaline phosphatase were restored to normal or greatly reduced in treated rats, indicating normalization of liver function. Non-viral insulin minicircle DNA-based TA1m mediated glucose-dependent insulin production in liver may represent a safe and promising approach to treat T1DM.

  15. Involvement of Gaba and Cannabinoid Receptors in Central Food Intake Regulation in Neonatal Layer Chicks: Role of CB1 and Gabaa Receptors

    Directory of Open Access Journals (Sweden)

    M Zendehdel

    Full Text Available ABSTRACT Feeding behavior is regulated via a complex network which interacts via diverse signals from central and peripheral tissues. Endocannabinoids modulate release of GABA in a variety of regions of the central nervous system. Endocannabinoids and GABAergic system have an important role in the central regulation of appetite. Thus, the present study examines the possible interaction of central canabinoidergic and GABAergic systems on food intake in 3-h food-deprived (FD3 neonatal layer-type chicks. The results of this study showed that intracerebroventricular (ICV injection of 2-AG (2-Arachidonoylglycerol, selective CB1 receptors agonist, 2µg significantly increased food intake and this effect of 2-AG was attenuated by Picrotoxin (GABAA antagonist, 0.5µg (P0.05. Also, hyperphagic effect of CB65 (CB2 receptors agonist, 1.25µg was not affected by Picrotoxin or CGP54626 (p>0.05. Moreover, the food intake of chicks was significantly increased by ICV injection of GABAA agonist (Gaboxadol, 0.2 µg and SR141716A (CB1 receptors antagonist, 6.25µg significantly decreased Gaboxadol-induced hyperphagia (P0.05. These data showed there might be an interaction between central cannabinoidergic and GABAergic systems via CB1 and GABAA receptors in control of food intake in neonatal layer chicks.

  16. The GABAA Antagonist DPP-4-PIOL Selectively Antagonises Tonic over Phasic GABAergic Currents in Dentate Gyrus Granule Cells

    DEFF Research Database (Denmark)

    Boddum, Kim; Frølund, Bente; Kristiansen, Uffe

    2014-01-01

    that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent...

  17. The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat α1β2γ2L GABAA receptor

    Science.gov (United States)

    Li, P; Akk, G

    2008-01-01

    Background and purpose: Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABAA receptors in the brain. In this study, we have examined the modulation of the common brain GABAA receptor subtype by fipronil and its major metabolite, fipronil sulphone. Experimental approach: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat α1β2γ2L GABAA receptors. Key results: The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The α1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the α1β2γ2L receptor. Conclusions and implications: We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain. PMID:18660823

  18. The sedating antidepressant trazodone impairs sleep-dependent cortical plasticity.

    Directory of Open Access Journals (Sweden)

    Sara J Aton

    2009-07-01

    Full Text Available Recent findings indicate that certain classes of hypnotics that target GABA(A receptors impair sleep-dependent brain plasticity. However, the effects of hypnotics acting at monoamine receptors (e.g., the antidepressant trazodone on this process are unknown. We therefore assessed the effects of commonly-prescribed medications for the treatment of insomnia (trazodone and the non-benzodiazepine GABA(A receptor agonists zaleplon and eszopiclone in a canonical model of sleep-dependent, in vivo synaptic plasticity in the primary visual cortex (V1 known as ocular dominance plasticity.After a 6-h baseline period of sleep/wake polysomnographic recording, cats underwent 6 h of continuous waking combined with monocular deprivation (MD to trigger synaptic remodeling. Cats subsequently received an i.p. injection of either vehicle, trazodone (10 mg/kg, zaleplon (10 mg/kg, or eszopiclone (1-10 mg/kg, and were allowed an 8-h period of post-MD sleep before ocular dominance plasticity was assessed. We found that while zaleplon and eszopiclone had profound effects on sleeping cortical electroencephalographic (EEG activity, only trazodone (which did not alter EEG activity significantly impaired sleep-dependent consolidation of ocular dominance plasticity. This was associated with deficits in both the normal depression of V1 neuronal responses to deprived-eye stimulation, and potentiation of responses to non-deprived eye stimulation, which accompany ocular dominance plasticity.Taken together, our data suggest that the monoamine receptors targeted by trazodone play an important role in sleep-dependent consolidation of synaptic plasticity. They also demonstrate that changes in sleep architecture are not necessarily reliable predictors of how hypnotics affect sleep-dependent neural functions.

  19. The effects of serotonin1A receptor on female mice body weight and food intake are associated with the differential expression of hypothalamic neuropeptides and the GABAA receptor.

    Science.gov (United States)

    Butt, Isma; Hong, Andrew; Di, Jing; Aracena, Sonia; Banerjee, Probal; Shen, Chang-Hui

    2014-10-01

    Both common eating disorders anorexia nervosa and bulimia nervosa are characteristically diseases of women. To characterize the role of the 5-HT1A receptor (5-HT1A-R) in these eating disorders in females, we investigated the effect of saline or 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) treatment on feeding behavior and body weight in adult WT female mice and in adult 5-HT1A-R knockout (KO) female mice. Our results showed that KO female mice have lower food intake and body weight than WT female mice. Administration of 8-OH-DPAT decreased food intake but not body weight in WT female mice. Furthermore, qRT-PCR was employed to analyze the expression levels of neuropeptides, γ-aminobutyric acid A receptor subunit β (GABAA β subunits) and glutamic acid decarboxylase in the hypothalamic area. The results showed the difference in food intake between WT and KO mice was accompanied by differential expression of POMC, CART and GABAA β2, and the difference in body weight between WT and KO mice was associated with significantly different expression levels of CART and GABAA β2. As such, our data provide new insight into the role of 5-HT1A-R in both feeding behavior and the associated expression of neuropeptides and the GABAA receptor. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Conglutinin exhibits a complement-dependent enhancement of the respiratory burst of phagocytes stimulated by E. coli

    DEFF Research Database (Denmark)

    Friis, P; Svehag, S E; Andersen, Ove

    1991-01-01

    . Conglutinin enhances, in a dose-dependent manner, the respiratory burst of spleen cells stimulated with serum-opsonized Escherichia coli. The enhancement was only demonstrable in the presence of a functional complement system. The conglutinin-mediated enhancement of the respiratory burst was inhibited...

  1. B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

    Directory of Open Access Journals (Sweden)

    Larimore Kevin

    2012-06-01

    Full Text Available Abstract Background The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. Results We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. Conclusions Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T cell-dependent antibody responses.

  2. First direct electron microscopic visualization of a tight spatial coupling between GABAA-receptors and voltage-sensitive calcium channels

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Belhage, B; Schousboe, A

    1992-01-01

    Using cerebellar granule neurons in culture it was demonstrated that exposure of the cells to the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) leads to an increase in the number of voltage-gated calcium channels as revealed by quantitative preembedding indirect imm...

  3. Cu(II) bis(thiosemicarbazone) radiopharmaceutical binding to serum albumin: further definition of species dependence and associated substituent effects

    International Nuclear Information System (INIS)

    Basken, Nathan E.; Green, Mark A.

    2009-01-01

    Introduction: The pyruvaldehyde bis(N 4 -methylthiosemicarbazonato)copper(II) (Cu-PTSM) and diacetyl bis(N 4 -methylthiosemicarbazonato)copper(II) (Cu-ATSM) radiopharmaceuticals exhibit strong, species-dependent binding to the IIA site of human serum albumin (HSA), while the related ethylglyoxal bis(thiosemicarbazonato)copper(II) (Cu-ETS) radiopharmaceutical appears to exhibit only nonspecific binding to HSA and animal serum albumins. Methods: To further probe the structural basis for the species dependence of this albumin binding interaction, we examined protein binding of these three radiopharmaceuticals in solutions of albumin and/or serum from a broader array of mammalian species (rat, sheep, donkey, rabbit, cow, pig, dog, baboon, mouse, cat and elephant). We also evaluated the albumin binding of several copper(II) bis(thiosemicarbazone) chelates offering more diverse substitution of the ligand backbone. Results: Cu-PTSM and Cu-ATSM exhibit a strong interaction with HSA that is not apparent with the albumins of other species, while the binding of Cu-ETS to albumin is much less species dependent. The strong interaction of Cu-PTSM with HSA does not appear to simply correlate with variation, relative to the animal albumins, of a single amino acid lining HSA's IIA site. Those agents that selectively interact with HSA share the common feature of only methyl or hydrogen substitution at the carbon atoms of the diimine fragment of the ligand backbone. Conclusions: The interspecies variations in albumin binding of Cu-PTSM and Cu-ATSM are not simply explained by unique amino acid substitutions in the IIA binding pocket of the serum albumins. However, the specific affinity for this region of HSA is disrupted when substituents bulkier than a methyl group appear on the imine carbons of the copper bis(thiosemicarbazone) chelate.

  4. Cu(II) bis(thiosemicarbazone) radiopharmaceutical binding to serum albumin: further definition of species dependence and associated substituent effects

    Energy Technology Data Exchange (ETDEWEB)

    Basken, Nathan E. [Division of Nuclear Pharmacy, Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907 (United States); Green, Mark A. [Division of Nuclear Pharmacy, Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907 (United States)], E-mail: magreen@purdue.edu

    2009-07-15

    Introduction: The pyruvaldehyde bis(N{sup 4}-methylthiosemicarbazonato)copper(II) (Cu-PTSM) and diacetyl bis(N{sup 4}-methylthiosemicarbazonato)copper(II) (Cu-ATSM) radiopharmaceuticals exhibit strong, species-dependent binding to the IIA site of human serum albumin (HSA), while the related ethylglyoxal bis(thiosemicarbazonato)copper(II) (Cu-ETS) radiopharmaceutical appears to exhibit only nonspecific binding to HSA and animal serum albumins. Methods: To further probe the structural basis for the species dependence of this albumin binding interaction, we examined protein binding of these three radiopharmaceuticals in solutions of albumin and/or serum from a broader array of mammalian species (rat, sheep, donkey, rabbit, cow, pig, dog, baboon, mouse, cat and elephant). We also evaluated the albumin binding of several copper(II) bis(thiosemicarbazone) chelates offering more diverse substitution of the ligand backbone. Results: Cu-PTSM and Cu-ATSM exhibit a strong interaction with HSA that is not apparent with the albumins of other species, while the binding of Cu-ETS to albumin is much less species dependent. The strong interaction of Cu-PTSM with HSA does not appear to simply correlate with variation, relative to the animal albumins, of a single amino acid lining HSA's IIA site. Those agents that selectively interact with HSA share the common feature of only methyl or hydrogen substitution at the carbon atoms of the diimine fragment of the ligand backbone. Conclusions: The interspecies variations in albumin binding of Cu-PTSM and Cu-ATSM are not simply explained by unique amino acid substitutions in the IIA binding pocket of the serum albumins. However, the specific affinity for this region of HSA is disrupted when substituents bulkier than a methyl group appear on the imine carbons of the copper bis(thiosemicarbazone) chelate.

  5. Identifying Urinary and Serum Exosome Biomarkers for Radiation Exposure Using a Data Dependent Acquisition and SWATH-MS Combined Workflow

    International Nuclear Information System (INIS)

    Kulkarni, Shilpa; Koller, Antonius; Mani, Kartik M.; Wen, Ruofeng; Alfieri, Alan; Saha, Subhrajit; Wang, Jian; Patel, Purvi; Bandeira, Nuno; Guha, Chandan

    2016-01-01

    Purpose: Early and accurate assessment of radiation injury by radiation-responsive biomarkers is critical for triage and early intervention. Biofluids such as urine and serum are convenient for such analysis. Recent research has also suggested that exosomes are a reliable source of biomarkers in disease progression. In the present study, we analyzed total urine proteome and exosomes isolated from urine or serum for potential biomarkers of acute and persistent radiation injury in mice exposed to lethal whole body irradiation (WBI). Methods and Materials: For feasibility studies, the mice were irradiated at 10.4 Gy WBI, and urine and serum samples were collected 24 and 72 hours after irradiation. Exosomes were isolated and analyzed using liquid chromatography mass spectrometry/mass spectrometry-based workflow for radiation exposure signatures. A data dependent acquisition and SWATH-MS combined workflow approach was used to identify significantly exosome biomarkers indicative of acute or persistent radiation-induced responses. For the validation studies, mice were exposed to 3, 6, 8, or 10 Gy WBI, and samples were analyzed for comparison. Results: A comparison between total urine proteomics and urine exosome proteomics demonstrated that exosome proteomic analysis was superior in identifying radiation signatures. Feasibility studies identified 23 biomarkers from urine and 24 biomarkers from serum exosomes after WBI. Urinary exosome signatures identified different physiological parameters than the ones obtained in serum exosomes. Exosome signatures from urine indicated injury to the liver, gastrointestinal, and genitourinary tracts. In contrast, serum showed vascular injuries and acute inflammation in response to radiation. Selected urinary exosomal biomarkers also showed changes at lower radiation doses in validation studies. Conclusions: Exosome proteomics revealed radiation- and time-dependent protein signatures after WBI. A total of 47 differentially secreted

  6. Identifying Urinary and Serum Exosome Biomarkers for Radiation Exposure Using a Data Dependent Acquisition and SWATH-MS Combined Workflow

    Energy Technology Data Exchange (ETDEWEB)

    Kulkarni, Shilpa [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Koller, Antonius [Proteomics Center, Stony Brook University School of Medicine, Stony Brook, New York (United States); Proteomics Shared Resource, Herbert Irving Comprehensive Cancer Center, New York, New York (United States); Mani, Kartik M. [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Wen, Ruofeng [Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, New York (United States); Alfieri, Alan; Saha, Subhrajit [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Wang, Jian [Center for Computational Mass Spectrometry, University of California, San Diego, California (United States); Department of Computer Science and Engineering, University of California, San Diego, California (United States); Patel, Purvi [Proteomics Shared Resource, Herbert Irving Comprehensive Cancer Center, New York, New York (United States); Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York (United States); Bandeira, Nuno [Center for Computational Mass Spectrometry, University of California, San Diego, California (United States); Department of Computer Science and Engineering, University of California, San Diego, California (United States); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California (United States); Guha, Chandan, E-mail: cguha@montefiore.org [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); and others

    2016-11-01

    Purpose: Early and accurate assessment of radiation injury by radiation-responsive biomarkers is critical for triage and early intervention. Biofluids such as urine and serum are convenient for such analysis. Recent research has also suggested that exosomes are a reliable source of biomarkers in disease progression. In the present study, we analyzed total urine proteome and exosomes isolated from urine or serum for potential biomarkers of acute and persistent radiation injury in mice exposed to lethal whole body irradiation (WBI). Methods and Materials: For feasibility studies, the mice were irradiated at 10.4 Gy WBI, and urine and serum samples were collected 24 and 72 hours after irradiation. Exosomes were isolated and analyzed using liquid chromatography mass spectrometry/mass spectrometry-based workflow for radiation exposure signatures. A data dependent acquisition and SWATH-MS combined workflow approach was used to identify significantly exosome biomarkers indicative of acute or persistent radiation-induced responses. For the validation studies, mice were exposed to 3, 6, 8, or 10 Gy WBI, and samples were analyzed for comparison. Results: A comparison between total urine proteomics and urine exosome proteomics demonstrated that exosome proteomic analysis was superior in identifying radiation signatures. Feasibility studies identified 23 biomarkers from urine and 24 biomarkers from serum exosomes after WBI. Urinary exosome signatures identified different physiological parameters than the ones obtained in serum exosomes. Exosome signatures from urine indicated injury to the liver, gastrointestinal, and genitourinary tracts. In contrast, serum showed vascular injuries and acute inflammation in response to radiation. Selected urinary exosomal biomarkers also showed changes at lower radiation doses in validation studies. Conclusions: Exosome proteomics revealed radiation- and time-dependent protein signatures after WBI. A total of 47 differentially secreted

  7. Separate and combined effects of the GABAA positive allosteric modulator diazepam and Δ⁹-THC in humans discriminating Δ⁹-THC.

    Science.gov (United States)

    Lile, Joshua A; Kelly, Thomas H; Hays, Lon R

    2014-10-01

    Our previous research suggested the involvement of γ-aminobutyric acid (GABA), in particular the GABAB receptor subtype, in the interoceptive effects of Δ(9)-tetrahydrocannabinol (Δ(9)-THC). The aim of the present study was to determine the potential involvement of the GABAA receptor subtype by assessing the separate and combined effects of the GABAA positive allosteric modulator diazepam and Δ(9)-THC using pharmacologically selective drug-discrimination procedures. Ten cannabis users learned to discriminate 30 mg oral Δ(9)-THC from placebo and then received diazepam (5 and 10mg), Δ(9)-THC (5, 15 and 30 mg) and placebo, alone and in combination. Self-report, task performance and physiological measures were also collected. Δ(9)-THC functioned as a discriminative stimulus, produced subjective effects typically associated with cannabinoids (e.g., High, Stoned, Like Drug) and elevated heart rate. Diazepam alone impaired performance on psychomotor performance tasks and increased ratings on a limited number of self-report questionnaire items (e.g., Any Effect, Sedated), but did not substitute for the Δ(9)-THC discriminative stimulus or alter the Δ(9)-THC discrimination dose-response function. Similarly, diazepam had limited impact on the other behavioral effects of Δ(9)-THC. These results suggest that the GABAA receptor subtype has minimal involvement in the interoceptive effects of Δ(9)-THC, and by extension cannabis, in humans. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat alpha1beta2gamma2L GABA(A) receptor.

    Science.gov (United States)

    Li, P; Akk, G

    2008-11-01

    Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABA(A) receptors in the brain. In this study, we have examined the modulation of the common brain GABA(A) receptor subtype by fipronil and its major metabolite, fipronil sulphone. Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat alpha1beta2gamma2L GABA(A) receptors. The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The alpha1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the alpha1beta2gamma2L receptor. We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain.

  9. Overexpression of PLK3 Mediates the Degradation of Abnormal Prion Proteins Dependent on Chaperone-Mediated Autophagy.

    Science.gov (United States)

    Wang, Hui; Tian, Chan; Sun, Jing; Chen, Li-Na; Lv, Yan; Yang, Xiao-Dong; Xiao, Kang; Wang, Jing; Chen, Cao; Shi, Qi; Shao, Qi-Xiang; Dong, Xiao-Ping

    2017-08-01

    Polo-like kinase 3 (PLK3) is the main cause of cell cycle reentry-related neuronal apoptosis which has been implicated in the pathogenesis of prion diseases. Previous work also showed the regulatory activity of exogenous PLK3 on the degradation of PrP (prion protein) mutants and pathogenic PrP Sc ; however, the precise mechanisms remain unknown. In this study, we identified that the overexpression of PLK3-mediated degradation of PrP mutant and PrP Sc was repressed by lysosome rather than by proteasomal and macroautophagy inhibitors. Core components of chaperone-mediated autophagy (CMA) effectors, lysosome-associated membrane protein type 2A (LAMP2a), and heat shock cognate protein 70 (Hsc70) are markedly decreased in the HEK293T cells expressing PrP mutant and scrapie-infected cell line SMB-S15. Meanwhile, PrP mutant showed ability to interact with LAMP2a and Hsc70. Overexpression of PLK3 sufficiently increased the cellular levels of LAMP2a and Hsc70, accompanying with declining the accumulations of PrP mutant and PrP Sc . The kinase domain (KD) of PLK3 was responsible for elevating LAMP2a and Hsc70. Knockdown of endogenous PLK3 enhanced the activity of macroautophagy in the cultured cells. Moreover, time-dependent reductions of LAMP2a and Hsc70 were also observed in the brain tissues of hamster-adapted scrapie agent 263K-infected hamsters, indicating an impairment of CMA during prion infection. Those data indicate that the overexpression of PLK3-mediated degradation of abnormal PrP is largely dependent on CMA pathway.

  10. Reexamination of mood-mediation hypothesis of background-music-dependent effects in free recall.

    Science.gov (United States)

    Isarida, Toshiko K; Kubota, Takayuki; Nakajima, Saki; Isarida, Takeo

    2017-03-01

    The present study reexamined the mood-mediation hypothesis for explaining background-music-dependent effects in free recall. Experiments 1 and 2 respectively examined tempo- and tonality-dependent effects in free recall, which had been used as evidence for the mood-mediation hypothesis. In Experiments 1 and 2, undergraduates (n = 75 per experiment) incidentally learned a list of 20 unrelated words presented one by one at a rate of 5 s per word and then received a 30-s delayed oral free-recall test. Throughout the study and test sessions, a piece of music was played. At the time of test, one third of the participants received the same piece of music with the same tempo or tonality as at study, one third heard a different piece with the same tempo or tonality, and one third heard a different piece with a different tempo or tonality. Note that the condition of the same piece with a different tempo or tonality was excluded. Furthermore, the number of sampled pieces of background music was increased compared with previous studies. The results showed neither tempo- nor tonality-dependent effects, but only a background-music-dependent effect. Experiment 3 (n = 40) compared the effects of background music with a verbal association task and focal music (only listening to musical selections) on the participants' moods. The results showed that both the music tempo and tonality influenced the corresponding mood dimensions (arousal and pleasantness). These results are taken as evidence against the mood-mediation hypothesis. Theoretical implications are discussed.

  11. Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus

    Science.gov (United States)

    Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S.; Wallner, Martin; Otis, Thomas S.

    2015-01-01

    The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit

  12. The stability of complement-mediated bactericidal activity in human serum against Salmonella.

    Directory of Open Access Journals (Sweden)

    Colette M O'Shaughnessy

    Full Text Available The complement cascade includes heat-labile proteins and care is required when handling serum in order to preserve its functional integrity. We have previously used a whole human serum bactericidal assay to show that antibody and an intact complement system are required in blood for killing of invasive isolates of Salmonella. The aim of the present study was to evaluate the conditions under which human serum can be stored and manipulated while maintaining complement integrity. Serum bactericidal activity against Salmonella was maintained for a minimum of 35 days when stored at 4°C, eight days at 22°C and 54 hours at 37°C. Up to three freeze-thaw cycles had no effect on the persistence of bactericidal activity and hemolytic complement assays confirmed no effect on complement function. Delay in the separation of serum for up to four days from clotted blood stored at 22°C did not affect bactericidal activity. Dilution of serum resulted in an increased rate of loss of bactericidal activity and so serum should be stored undiluted. These findings indicate that the current guidelines concerning manipulation and storage of human serum to preserve complement integrity and function leave a large margin for safety with regards to bactericidal activity against Salmonella. The study provides a scheme for determining the requirements for serum handling in relation to functional activity of complement in other systems.

  13. Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis

    Science.gov (United States)

    Calder, Elizabeth A.; Penhale, W. J.; McLeman, Dena; Barnes, E. W.; Irvine, W. J.

    1973-01-01

    In the presence of normal human lymphocytes, decomplemented sera from twentynine out of thirty-nine patients with Hashimoto thyroiditis caused significant lysis of thyroglobulin-coated chicken red blood cells, as estimated by the release of 51Cr; the mean% specific 51Cr release being 14·1 ± 1·9 (SEM). Serum from twenty-one control subjects studied concurrently caused no significant lysis of thyroglobulin-coated chicken red blood cells; the mean% specific 51Cr release being −1·6±0·7 (SEM). The degree of cytotoxicity correlated with the titre of thyroglobulin antibodies in the serum, determined by tanned red cell haemagglutination. The active component in the Hashimoto serum was localized in the 19S fraction, was unaffected by pre-absorption with anti-human IgM serum, but was neutralized by pre-absorption with anti-human IgG serum. These findings suggest that the cytotoxic activity of serum from patients with Hashimoto thyroiditis is due to the presence of thyroglobulin antibody of the IgG class in the form of complexes, either alone or with antigen. It is postulated that non-specific lymphocytes may play an important role in the pathogenesis of Hashimoto thyroiditis, being activated by the presence in the gland of thyroglobulin antibody, either alone or in the form of complexes attached to thyroid cells. PMID:4740445

  14. Activation of the human complement system by cholesterol-rich and pegylated liposomes - Modulation of cholesterol-rich liposome-mediated complement activation by elevated serum LDL and HDL levels

    DEFF Research Database (Denmark)

    Moghimi, S.M.; Hamad, I.; Bunger, R.

    2006-01-01

    level of S-protein-bound form of the terminal complex (SC5b-9). However, liposome-induced rise of SC5b-9 was significantly suppressed when serum HDL cholesterol levels increased by 30%. Increase of serum LDL to levels similar to that observed in heterozygous familial hypercholesterolemia also suppressed......Intravenously infused liposomes may induce cardiopulmonary distress in some human subjects, which is a manifestation of "complement activation-related pseudoallergy." We have now examined liposome-mediated complement activation in human sera with elevated lipoprotein (LDL and HDL) levels, since...... abnormal or racial differences in serum lipid profiles seem to modulate the extent of complement activation and associated adverse responses. In accordance with our earlier observations, cholesterol-rich (45 mol% cholesterol) liposomes activated human complement, as reflected by a significant rise in serum...

  15. Three cases of zolpidem dependence treated with fluoxetine: the serotonin hypothesis.

    Science.gov (United States)

    Liappas, Ioannis A; Malitas, Petros N; Dimopoulos, Nikolaos P; Gitsa, Olympia E; Liappas, Alexandros I; Nikolaou, Chrisoula K; Christodoulou, Georgios N

    2003-04-01

    Zolpidem is an imidazopyridine hypnotic that is believed to act selectively at alpha(1) subunit-containing gamma-aminobutyric acid type A (GABA(A)) receptors and thus to have minimal abuse and dependence potential. We present three cases of zolpidem abuse and dependence in which the drug was used not for sedation but for stimulation and anxiolysis. All of the patients were treated with fluoxetine (a selective serotonin reuptake inhibitor) and managed to discontinue the abuse and remain abstinent from the drug. The efficacy of this kind of medication on the abuse of a GABAergic agonist, in this case dependence on zolpidem, leads to a serotonergic and GABAergic system interaction hypothesis.

  16. ATP binding cassette G1-dependent cholesterol efflux during inflammation.

    Science.gov (United States)

    de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

    2011-02-01

    ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

  17. Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

    Science.gov (United States)

    Gonzalez, Deyarina; Hamidi, Nurul; Del Sol, Ricardo; Benschop, Joris J; Nancy, Thomas; Li, Chao; Francis, Lewis; Tzouros, Manuel; Krijgsveld, Jeroen; Holstege, Frank C P; Conlan, R Steven

    2014-02-18

    Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

  18. The influence of stress at puberty on mood and learning: Role of the α4βδ GABAA receptor

    Science.gov (United States)

    Smith, Sheryl S.

    2012-01-01

    It is well-known that the onset of puberty is associated with changes in mood as well as cognition. Stress can have an impact on these outcomes, which in many cases, can be more influential in females, suggesting that gender differences exist. The adolescent period is a vulnerable time for the onset of certain psychopathologies, including anxiety disorders, depression and eating disorders, which are also more prevalent in females. One factor which may contribute to stress-triggered anxiety at puberty is the GABAA receptor (GABAR), which is known to play a pivotal role in anxiety. Expression of α4βδ GABARs increases on the dendrites of CA1 pyramidal cells at the onset of puberty in the hippocampus, part of the limbic circuitry which governs emotion. This receptor is a sensitive target for the stress steroid THP (3α-OH-5[α]β-pregnan-20-one), which paradoxically reduces inhibition and increases anxiety during the pubertal period (~PND 35–44) of female mice in contrast to its usual effect to enhance inhibition and reduce anxiety. Spatial learning and synaptic plasticity are also adversely impacted at puberty, likely a result of increased expression of α4βδ GABARs on the dendritic spines of CA1 hippocampal pyramidal cells, which are essential for consolidation of memory. This review will focus on the role of these receptors in mediating behavioral changes at puberty. Stress-mediated changes in mood and cognition in early adolescence may have relevance for the expression of psychopathologies in adulthood. PMID:23079628

  19. Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

    Science.gov (United States)

    Bialuk, Izabela; Whitney, Stephen; Andresen, Vibeke; Florese, Ruth H; Nacsa, Janos; Cecchinato, Valentina; Valeri, Valerio W; Heraud, Jean-Michel; Gordon, Shari; Parks, Robyn Washington; Montefiori, David C; Venzon, David; Demberg, Thorsten; Guroff, Marjorie Robert-; Landucci, Gary; Forthal, Donald N; Franchini, Genoveffa

    2011-12-09

    The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection. Published by Elsevier Ltd.

  20. Human erythrocytes inhibit complement-mediated solubilization of immune complexes by human serum

    International Nuclear Information System (INIS)

    Dorval, B.L.

    1987-01-01

    The aim of this study was to develop an autologus human system to evaluate the effects of human erythrocytes on solubilization of immune complex precipitates (IC) by human serum. Incubation of IC with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed erythrocytes were added to human serum or guinea pig serum binding of IC to the erythrocyte occurred and IC solubilization was inhibited significantly (p <.025). Sheep erythrocytes did not bind IC or inhibit IC solubilization. To evaluate the role of human erythrocyte complement receptor (CR1) on these findings, human erythrocytes were treated with trypsin or anti-CR1 antibodies. Both treatments abrogated IC binding to human erythrocytes but did not affect the ability of the human erythrocyte to inhibit IC solubilization. Radioimmunoassay was used to measure C3, C4 and C5 activation in human serum after incubation with IC, human erythrocytes, human erythrocytes plus IC, whole blood or in whole blood plus IC

  1. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    International Nuclear Information System (INIS)

    Ding, L.; Wu, J.P.; Xu, G.; Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W.

    2014-01-01

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis

  2. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ding, L.; Wu, J.P. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China); Xu, G. [Fudan University, Jinshan Hospital, Center Laboratory, Shanghai, China, Center Laboratory, Jinshan Hospital, Fudan University, Shanghai (China); Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China)

    2014-05-09

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  3. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Directory of Open Access Journals (Sweden)

    L. Ding

    2014-06-01

    Full Text Available Current studies find that degenerated cartilage endplates (CEP of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  4. Allopregnanolone suppresses diabetes-induced neuropathic pain and motor deficit through inhibition of GABAA receptor down-regulation in the spinal cord of diabetic rats

    Directory of Open Access Journals (Sweden)

    Samira Afrazi

    2014-05-01

    Full Text Available Objective(s:Painful diabetic neuropathy is associated with hyperexcitability and hyperactivity of spinal cord neurons. However, its underlying pathophysiological mechanisms have not been fully clarified. Induction of excitatory/inhibitory neurotransmission imbalance at the spinal cord seems to account for the abnormal neuronal activity in diabetes. Protective properties of neurosteroids have been demonstrated in numerous cellular and animal models of neurodegeneration. Materials and Methods: Here, the protective effects of allopregnanolone, a neurosteroid were investigated in an in vivo model of diabetic neuropathy. The tail-flick test was used to assess the nociceptive threshold. Diabetes was induced by injection of 50 mg/kg (IP streptozotocin. Seven weeks after the induction of diabetes, the dorsal half of the lumbar spinal cord was assayed for the expression of γ2 subunit of GABAA receptor using semiquantitative RT-PCR. Results: The data shows that allopregnanolone (5 and 20 mg/kg markedly ameliorated diabetes-induced thermal hyperalgesia and motor deficit. The weights of diabetic rats that received 5 and 20 mg/kg allopregnanolone did not significantly reduce during the time course of study. Furthermore, this neurosteroid could inhibit GABAA receptor down-regulation induced by diabetes in the rat spinal cord. Conclusion: The data revealed that allopregnanolone has preventive effects against hyperglycemic-induced neuropathic pain and motor deficit which are related to the inhibition of GABAA receptor down-regulation.

  5. A Review of the Updated Pharmacophore for the Alpha 5 GABA(A Benzodiazepine Receptor Model

    Directory of Open Access Journals (Sweden)

    Terry Clayton

    2015-01-01

    Full Text Available An updated model of the GABA(A benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1 which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM, SH-053-2′F-R-CH3 (2, has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A receptors.

  6. Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors.

    Directory of Open Access Journals (Sweden)

    Alexis M Ziemba

    Full Text Available Pentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range.Using oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep "notch" approach, and used these results to correct steady-state direct activation for inhibition.Combining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA.Our novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites.

  7. The role of HSP70 in mediating age-dependent mortality in sepsis

    Science.gov (United States)

    McConnell, Kevin W.; Fox, Amy C.; Clark, Andrew T.; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A.; Farris, Alton B.; Buchman, Timothy G.; Hunt, Clayton R.; Coopersmith, Craig M.

    2011-01-01

    Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6–12week old) and aged (16–17 month old) HSP70−/− and wild type (WT) mice to determine if HSP70 modulated outcome in an age-dependent fashion. Young HSP70−/− and WT mice subjected to cecal ligation and puncture (CLP), Pseudomonas aeruginosa pneumonia or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70−/− mice than aged WT mice subjected to CLP (p=0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared to WT mice, aged septic HSP70−/− mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70−/−mice had increased systemic levels of TNF-α, IL-6, IL-10 and IL-1β compared to WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation. PMID:21296977

  8. Extracellular pH modulates GABAergic neurotransmission in rat hypothalamus.

    Science.gov (United States)

    Chen, Z L; Huang, R Q

    2014-06-20

    Changes in extracellular pH have a modulatory effect on GABAA receptor function. It has been reported that pH sensitivity of the GABA receptor is dependent on subunit composition and GABA concentration. Most of previous investigations focused on GABA-evoked currents, which only reflect the postsynaptic receptors. The physiological relevance of pH modulation of GABAergic neurotransmission is not fully elucidated. In the present studies, we examined the influence of extracellular pH on the GABAA receptor-mediated inhibitory neurotransmission in rat hypothalamic neurons. The inhibitory postsynaptic currents (IPSCs), tonic currents, and the GABA-evoked currents were recorded with whole-cell patch techniques on the hypothalamic slices from Sprague-Dawley rats at 15-26 postnatal days. The amplitude and frequency of spontaneous GABA IPSCs were significantly increased while the external pH was changed from 7.3 to 8.4. In the acidic pH (6.4), the spontaneous GABA IPSCs were reduced in amplitude and frequency. The pH induced changes in miniature GABA IPSCs (mIPSCs) similar to that in spontaneous IPSCs. The pH effect on the postsynaptic GABA receptors was assessed with exogenously applied varying concentrations of GABA. The tonic currents and the currents evoked by sub-saturating concentration of GABA ([GABA]) (10 μM) were inhibited by acidic pH and potentiated by alkaline pH. In contrast, the currents evoked by saturating [GABA] (1mM) were not affected by pH changes. We also investigated the influence of pH buffers and buffering capacity on pH sensitivity of GABAA receptors on human recombinant α1β2γ2 GABAA receptors stably expressed in HEK 293 cells. The pH influence on GABAA receptors was similar in HEPES- and MES-buffered media, and not dependent on protonated buffers, suggesting that the observed pH effect on GABA response is a specific consequence of changes in extracellular protons. Our data suggest that the hydrogen ions suppress the GABAergic neurotransmission

  9. Serum magnesium and the risk of prediabetes: a population-based cohort study.

    Science.gov (United States)

    Kieboom, Brenda C T; Ligthart, Symen; Dehghan, Abbas; Kurstjens, Steef; de Baaij, Jeroen H F; Franco, Oscar H; Hofman, Albert; Zietse, Robert; Stricker, Bruno H; Hoorn, Ewout J

    2017-05-01

    Previous studies have found an association between serum magnesium and incident diabetes; however, this association may be due to reverse causation, whereby diabetes may induce urinary magnesium loss. In contrast, in prediabetes (defined as impaired fasting glucose), serum glucose levels are below the threshold for urinary magnesium wasting and, hence, unlikely to influence serum magnesium levels. Thus, to study the directionality of the association between serum magnesium levels and diabetes, we investigated its association with prediabetes. We also investigated whether magnesium-regulating genes influence diabetes risk through serum magnesium levels. Additionally, we quantified the effect of insulin resistance in the association between serum magnesium levels and diabetes risk. Within the population-based Rotterdam Study, we used Cox models, adjusted for age, sex, lifestyle factors, comorbidities, kidney function, serum levels of electrolytes and diuretic use, to study the association between serum magnesium and prediabetes/diabetes. In addition, we performed two mediation analyses: (1) to study if common genetic variation in eight magnesium-regulating genes influence diabetes risk through serum magnesium levels; and (2) to quantify the proportion of the effect of serum magnesium levels on diabetes that is mediated through insulin resistance (quantified by HOMA-IR). A total of 8555 participants (mean age, 64.7 years; median follow-up, 5.7 years) with normal glucose levels (mean ± SD: 5.46 ± 0.58 mmol/l) at baseline were included. A 0.1 mmol/l decrease in serum magnesium level was associated with an increase in diabetes risk (HR 1.18 [95% CI 1.04, 1.33]), confirming findings from previous studies. Of interest, a similar association was found between serum magnesium levels and prediabetes risk (HR 1.12 [95% CI 1.01, 1.25]). Genetic variation in CLDN19, CNNM2, FXYD2, SLC41A2, and TRPM6 significantly influenced diabetes risk (p prediabetes 13.4% was

  10. Electrical Stimulation at the ST36 Acupoint Protects against Sepsis Lethality and Reduces Serum TNF Levels through Vagus Nerve- and Catecholamine-Dependent Mechanisms

    Directory of Open Access Journals (Sweden)

    Albino Villegas-Bastida

    2014-01-01

    Full Text Available Electrical vagus nerve (VN stimulation during sepsis attenuates tumor necrosis factor (TNF production through the cholinergic anti-inflammatory pathway, which depends on the integrity of the VN and catecholamine production. To characterize the effect of electroacupuncture at ST36 (EA-ST36 on serum TNF, IL-6, nitrite, and HMGB1 levels and survival rates, based on VN integrity and catecholamine production, a sepsis model was induced in rats using cecal ligation and puncture (CLP. The septic rats were subsequently treated with EA-ST36 (CLP+ST36, and serum samples were collected and analyzed for cytokines levels. The serum TNF, IL-6, nitrite, and HMGB1 levels in the CLP+ST36 group were significantly lower compared with the group without treatment, the survival rates were significantly higher (P<0.05, and the acute organ injury induced by CLP was mitigated by EA-ST36; however, when subdiaphragmatic vagotomy was performed, the serum levels of TNF in the CLP+ST36 group did not show a significant difference compared with the group without electrostimulation, and, similarly, no significant difference in serum TNF levels was found under the pharmacological blockade of catecholamines. These results suggest that in rats with CLP sepsis models EA-ST36 reduces serum TNF levels through VN- and atecholamine-dependent mechanisms.

  11. Dose- and time-dependence of the host-mediated response to paclitaxel therapy: a mathematical modeling approach.

    Science.gov (United States)

    Benguigui, Madeleine; Alishekevitz, Dror; Timaner, Michael; Shechter, Dvir; Raviv, Ziv; Benzekry, Sebastien; Shaked, Yuval

    2018-01-05

    It has recently been suggested that pro-tumorigenic host-mediated processes induced in response to chemotherapy counteract the anti-tumor activity of therapy, and thereby decrease net therapeutic outcome. Here we use experimental data to formulate a mathematical model describing the host response to different doses of paclitaxel (PTX) chemotherapy as well as the duration of the response. Three previously described host-mediated effects are used as readouts for the host response to therapy. These include the levels of circulating endothelial progenitor cells in peripheral blood and the effect of plasma derived from PTX-treated mice on migratory and invasive properties of tumor cells in vitro . A first set of mathematical models, based on basic principles of pharmacokinetics/pharmacodynamics, did not appropriately describe the dose-dependence and duration of the host response regarding the effects on invasion. We therefore provide an alternative mathematical model with a dose-dependent threshold, instead of a concentration-dependent one, that describes better the data. This model is integrated into a global model defining all three host-mediated effects. It not only precisely describes the data, but also correctly predicts host-mediated effects at different doses as well as the duration of the host response. This mathematical model may serve as a tool to predict the host response to chemotherapy in cancer patients, and therefore may be used to design chemotherapy regimens with improved therapeutic outcome by minimizing host mediated effects.

  12. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David

    2007-01-01

    Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method...... to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor......, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain...

  13. Associations among serum pro- and anti-inflammatory cytokines, metabolic mediators, body condition, and uterine disease in postpartum dairy cows.

    Science.gov (United States)

    Kasimanickam, Ramanathan K; Kasimanickam, Vanmathy R; Olsen, Jesse R; Jeffress, Erin J; Moore, Dale A; Kastelic, John P

    2013-11-09

    Adipose tissue is an active endocrine organ which secretes a wide range of hormones and protein factors, collectively termed adipokines. Adipokines affect appetite and satiety, glucose and lipid metabolism, inflammation and immune functions. The objectives were to evaluate serum concentrations of adipokines (adiponectin, leptin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6) in lactating dairy cows with postpartum uterine inflammatory conditions (metritis, clinical endometritis or subclinical endometritis) and in cows experiencing loss of body condition, and to assess the relationship of adipokines and body condition loss in the establishment of persistent uterine inflammatory conditions. Lactating multiparous Holstein cows (N = 40), with body condition scores (BCS) from 2 to 4 (eight cows for each 0.5 score increment) were enrolled. Body condition was monitored for all cows weekly for 7 weeks post calving; cows with uterine inflammatory conditions were also re-evaluated 2 weeks later. Blood samples were collected from 1 week prior to calving to 7 weeks after calving for determination of serum concentrations of adipokines, insulin and insulin like growth factor (IGF)-1. Cows with metritis or clinical endometritis had higher serum concentrations of adiponectin, leptin, TNF-alpha, IL-1beta and IL-6 compared to normal cows (P cows with subclinical endometritis compared to normal cows (P insulin and IGF-1 concentrations were lower in cows with metritis or clinical endometritis. Cows with low BCS (2 and 2.5) had significantly higher adiponectin, TNF-alpha, IL-1beta and IL-6 than those with high BCS (3 to 4). Cows with persistent uterine inflammatory conditions had higher adiponectin, leptin TNF-alpha, IL-1beta and IL-6 and insulin compared to normal and spontaneously recovered cows, except for IGF-1 (P insulin, and IGF-1 had significant associations with BCS categories (low vs. high) and postpartum uterine inflammatory conditions. Perhaps loss of

  14. Kampo medicine: Evaluation of the pharmacological activity of 121 herbal drugs on GABA(A and 5 HT3A receptors

    Directory of Open Access Journals (Sweden)

    Katrin M Hoffmann

    2016-07-01

    Full Text Available Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM. During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and its constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, we performed a broad screening of Kampo remedies to look for pharmacologically relevant 5 HT3A and GABA(A receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness or insomnia. Therefore, we analyzed the pharmacological effects of 121 herbal drugs from Kampo medicine as ethanol tinctures on heterologously expressed 5 HT3A and GABA(A receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix and Leonurus japonicus (herba were the most effective inhibitory compounds on the 5 HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5 HT3A receptor antagonist. We also identified several potentiating herbs (e.g., Magnolia officinalis (cortex, Syzygium aromaticum (flos and Panax ginseng (radix for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects, for instance Salvia miltiorrhiza (radix were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing a better understanding of their physiological effects and clinical applications.

  15. GABAA receptor endocytosis in the basolateral amygdala is critical to the reinstatement of fear memory measured by fear-potentiated startle.

    Science.gov (United States)

    Lin, Hui-Ching; Tseng, Yu-Chou; Mao, Sheng-Chun; Chen, Po-See; Gean, Po-Wu

    2011-06-15

    Reinstatement represents a phenomenon that may be used to model the effects of retraumatization observed in patients with posttraumatic stress disorder (PTSD). In this study, we found intraperitoneal injection of the β-adrenergic receptor antagonist propranolol (10 mg/kg) 1 h before reinstatement training attenuated reinstatement of fear memory in rats. Conversely, reinstatement was facilitated by intra-amygdalar administration of β-adrenergic receptor agonist isoproterenol (Iso; 2 μg per side) 30 min before reinstatement training. The frequency and amplitude of the miniature IPSC (mIPSC) and the surface expression of the β3 and γ2 subunits of the GABA(A) receptor (GABA(A)R) were significantly lower in reinstated than in extinction rats, whereas the AMPA/NMDA ratio and the surface expression of GluR1 and GluR2 in the amygdala did not differ between groups. In amygdala slices, Iso-induced decrease in the surface β3 subunit of GABA(A) receptor was blocked by a Tat-conjugated dynamin function-blocking peptide (Tat-P4) pretreatment (10 μm for 30 min). By contrast, Tat-scramble peptide had no effect. Intravenous injection (3 μmol/kg) or intra-amygdalar infusion (30 pmol per side) of Tat-P4 interfered with reinstatement. Reinstatement increased the association between protein phosphatase 2A (PP2A) and the β3 subunit of the GABA(A)R, which was abolished by PP1/PP2A inhibitors okadaic acid and calyculin A. These results suggest the involvement of β-adrenergic receptor activation and GABA(A) receptor endocytosis in the amygdala for the reinstatement in fear memory.

  16. The GABAA receptor complex in hepatic encephalopathy. Autoradiographic evidence for the presence of elevated levels of a benzodiazepine receptor ligand

    Energy Technology Data Exchange (ETDEWEB)

    Basile, A.S.; Ostrowski, N.L.; Gammal, S.H.; Jones, E.A.; Skolnick, P. (National Institutes of Health, Bethesda, MD (USA))

    1990-02-01

    Autoradiographic analysis was used to examine radioligand binding to benzodiazepine (BZ) and GABAA receptors in the brains of rabbits with hepatic encephalopathy (HE). Thin sections of whole brain from normal rabbits and rabbits with HE were mounted on slides and subdivided into two groups. One group was washed before incubation with radioligand, while the second group was not prewashed. (3H)Flunitrazepam binding to BZ receptors was decreased by 22% to 42% (p less than 0.05) in the cerebral cortex, superior and inferior colliculi, and cerebellum of unwashed sections from rabbits with HE compared to all other groups. The binding of (3H)Ro 15-1788 to unwashed sections from rabbits with HE was reduced by a similar degree (18% to 37%, p less than 0.05) in the cerebral cortex, hippocampus, superior colliculus, and cerebellar cortex. Incubation of sections with the GABA-mimetic muscimol and NaCl produced an additional decrease in (3H)flunitrazepam binding to the cortex and hippocampus (25% to 31%, p less than 0.05) in unwashed HE rabbit brain, but increased radioligand binding (27% to 71%, p less than 0.05) to several regions in control rabbits. No changes in radioligand binding to either GABAA or peripheral benzodiazepine receptors was observed between HE and control rabbit sections. These findings are consistent with previous electrophysiologic and neurochemical observations indicating no significant changes in either the function or density of GABAA or BZ receptors in this model of HE. Further, they indicate that a reversible BZ receptor ligand with agonist properties is present in the brain in HE. This substance may contribute to the enhancement of GABAergic tone observed in this syndrome.

  17. SGLT1-mediated transport in Caco-2 cells is highly dependent on cell bank origin

    DEFF Research Database (Denmark)

    Steffansen, B; Pedersen, Maria; Laghmoch, A M

    2017-01-01

    The Caco-2 cell line is a well-established in vitro model for studying transport phenomena for prediction of intestinal nutrient and drug absorption. However, for substances depending on transporters such predictions are complicated due to variable transporter expression and limited knowledge about...... transporter function during multiple cell passaging and cell thawings. In the case of SGLT1, a key transporter of oral absorption of D-glucose, one reason for compromised prediction could be inadequate expression of SGLT1 in Caco-2 cells and thereby limited sensitivity in the determination of SGLT1-mediated...... permeability (PSGLT1). Here, the objective was to characterize and compare SGLT1-mediated uptake in Caco-2 cells obtained from different cell banks. SGLT1-mediated uptake of the standard SGLT1 substrate, α-MDG, in Caco-2 cells was shown to be highly dependent on cell bank origin. The most robust and reliable...

  18. The wavelength dependence of gold nanorod-mediated optical breakdown during infrared ultrashort pulses

    Energy Technology Data Exchange (ETDEWEB)

    Davletshin, Yevgeniy R.; Kumaradas, J. Carl [Department of Physics, Ryerson University, Toronto, ON (Canada)

    2017-04-15

    This paper investigates the wavelength dependence of the threshold of gold nanorod-mediated optical breakdown during picosecond and femtosecond near infrared optical pulses. It was found that the wavelength dependence in the picosecond regime is governed solely by the changes of a nanorod's optical properties. On the other hand, the optical breakdown threshold during femtosecond pulse exposure falls within one of two regimes. When the ratio of the maximum electric field from the outside to the inside of the nanorod is less then 7 (the absorption regime) the seed electrons are initiated by photo-thermal emission, and the wavelength dependence in the threshold of optical breakdown is the result of optical properties of the nanoparticle. When the ratio is greater than 7 (the near-field regime) more seed electrons are initiated by multiphoton ionization, and the wavelength dependence of the threshold of optical breakdown results from a combination of nanorod's optical properties and transitions in the order of multiphoton ionization. The findings of this study can guide the design of nanoparticle based optical breakdown applications. This analysis also deepens the understanding of nanoparticle-mediated laser induced breakdown for picosecond and femtosecond pulses at near infrared wavelengths. (copyright 2017 by WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  19. Epidermal growth factor-mediated effects on equine vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Grosenbaugh, D.A.; Amoss, M.S.; Hood, D.M.; Morgan, S.J.; Williams, J.D.

    1988-01-01

    Epidermal growth factor (EGF) receptor binding kinetics and EGF-mediated stimulation of DNA synthesis and cellular proliferation were studied in cultured vascular smooth muscle cells (VSMC) from the equine thoracic aorta. Binding studies, using murine 125 I-labeled EGF, indicate the presence of a single class of high-affinity binding sites, with an estimated maximal binding capacity of 5,800 sites/cells. EGF stimulated [ 3 H]thymidine uptake in confluent quiescent monolayers in a dose-dependent fashion, half-maximal stimulation occurring at 7.5 x 10 -11 M. Likewise, EGF-mediated cellular proliferation was dose dependent under reduced serum concentrations. Equine VSMC contain specific receptors for EGF, and EGF can stimulate DNA synthesis and proliferation in these cultured cells, which suggests that EGF may participate in the proliferative changes observed in equine distal digital peripheral vascular disease

  20. Value of serum tenascin-C in patients with acute myocardial infarction

    African Journals Online (AJOL)

    Rania Gaber

    2015-10-09

    Oct 9, 2015 ... Aliquots of serum were delivered into Eppendorf tubes. Serum samples have been .... Tenascin-C induced by aldosterone-mediated inflammation. J · Cardiovasc .... nosis estimation and targeted therapy. Cell Adh Migr 2015 ...

  1. Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA.

    Science.gov (United States)

    Yang, J P; Huang, L

    1997-09-01

    Since cationic liposome was first developed as a lipofection reagent, a drawback has been noted in that the efficiency of lipofection decreases dramatically after addition of serum to the lipofection medium. This drawback hampers the application of cationic liposome for systematic delivery of genes. In the present studies, we found that the effect of serum on DC-chol liposome-mediated lipofection is dependent on the charge ratio of liposome to DNA. Serum inhibited lipofection activity of the lipoplex at low charge ratios, whereas it enhanced the lipofection activity at high charge ratios. This phenomenon was observed using DOTAP/DOPE but not lipofectamine. Measurement of cellular association of DNA showed that serum could reduce the binding of lipoplex to cells at all tested charge ratios, i.e. 0-10.6. Removal of negatively charged proteins from serum by DEAE Sephacel column abolished the inhibitory effect of serum on lipofection. The fraction contained only negatively charged serum proteins which strongly inhibited lipofection at low charge ratios but not at higher charge ratios. Furthermore, preincubation of serum with positively charged polylysine, which neutralized negatively charged serum proteins, eliminated the inhibitory effect of serum on lipofection. In summary, inactivation of cationic liposome by serum is due to negatively charged serum proteins and it can be overcome by increasing charge ratio of cationic liposome-DNA lipoplexes or by neutralizing the serum with polylysine.

  2. Nanoparticle Delivery of Artesunate Enhances the Anti-tumor Efficiency by Activating Mitochondria-Mediated Cell Apoptosis

    Science.gov (United States)

    Liu, Rui; Yu, Xiwei; Su, Chang; Shi, Yijie; Zhao, Liang

    2017-06-01

    Artemisinin and its derivatives were considered to exert a broad spectrum of anti-cancer activities, and they induced significant anti-cancer effects in tumor cells. Artemisinin and its derivatives could be absorbed quickly, and they were widely distributed, selectively killing tumor cells. Since low concentrations of artesunate primarily depended on oncosis to induce cell death in tumor cells, its anti-tumor effects were undesirable and limited. To obtain better anti-tumor effects, in this study, we took advantage of a new nanotechnology to design novel artesunate-loaded bovine serum albumin nanoparticles to achieve the mitochondrial accumulation of artesunate and induce mitochondrial-mediated apoptosis. The results showed that when compared with free artesunate's reliance on oncotic death, artesunate-loaded bovine serum albumin nanoparticles showed higher cytotoxicity and their significant apoptotic effects were induced through the distribution of artesunate in the mitochondria. This finding indicated that artesunate-loaded bovine serum albumin nanoparticles damaged the mitochondrial integrity and activated mitochondrial-mediated cell apoptosis by upregulating apoptosis-related proteins and facilitating the rapid release of cytochrome C.

  3. Zolpidem reduces the blood oxygen level-dependent signal during visual system stimulation

    OpenAIRE

    Licata, Stephanie C.; Lowen, Steven B.; Trksak, George H.; MacLean, Robert R.; Lukas, Scott E.

    2011-01-01

    Zolpidem is a short-acting imidazopyridine hypnotic that binds at the benzodiazepine binding site on specific GABAA receptors to enhance fast inhibitory neurotransmission. The behavioral and receptor pharmacology of zolpidem has been studied extensively, but little is known about its neuronal substrates in vivo. In the present within-subject, double-blind, and placebo-controlled study, blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) at 3 Tesla was used to assess...

  4. Hypocretin and GABA interact in the pontine reticular formation to increase wakefulness.

    Science.gov (United States)

    Brevig, Holly N; Watson, Christopher J; Lydic, Ralph; Baghdoyan, Helen A

    2010-10-01

    Hypocretin-1/orexin A administered directly into the oral part of rat pontine reticular formation (PnO) causes an increase in wakefulness and extracellular gamma-aminobutyric acid (GABA) levels. The receptors in the PnO that mediate these effects have not been identified. Therefore, this study tested the hypothesis that the increase in wakefulness caused by administration of hypocretin-1 into the PnO occurs via activation of GABAA receptors and hypocretin receptors. Within/between subjects. University of Michigan. Twenty-three adult male Crl:CD*(SD) (Sprague Dawley) rats. Microinjection of hypocretin-1, bicuculline (GABAA receptor antagonist), SB-334867 (hypocretin receptor-1 antagonist), and Ringer solution (vehicle control) into the PnO. Hypocretin-1 caused a significant concentration-dependent increase in wakefulness and decrease in rapid eye movement (REM) sleep and non-REM (NREM) sleep. Coadministration of SB-334867 and hypocretin-1 blocked the hypocretin-1-induced increase in wakefulness and decrease in both the NREM and REM phases of sleep. Coadministration of bicuculline and hypocretin-1 blocked the hypocretin-1-induced increase in wakefulness and decrease in NREM sleep caused by hypocretin-1. The increase in wakefulness caused by administering hypocretin-1 to the PnO is mediated by hypocretin receptors and GABAA receptors in the PnO. These results show for the first time that hypocretinergic and GABAergic transmission in the PnO can interact to promote wakefulness.

  5. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

    Science.gov (United States)

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-02-08

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

  6. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor γ2 subunit

    Science.gov (United States)

    Kittler, Josef T.; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R.; McAinsh, Kristina; Arancibia-Carcamo, I. Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J.

    2008-01-01

    The regulation of the number of γ2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface γ2-subunit-containing GABAARs is regulated. Here, we identify a γ2-subunit-specific Yxxφ-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for γ2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxφ motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that γ2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition. PMID:18305175

  7. Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

    Science.gov (United States)

    Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

    2010-07-30

    Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

  8. Characterization of the effect of serum and chelating agents on Staphylococcus aureus biofilm formation; chelating agents augment biofilm formation through clumping factor B

    Science.gov (United States)

    Abraham, Nabil Mathew

    Staphylococcus aureus is the causative agent of a diverse array of acute and chronic infections, and some these infections, including infective endocarditis, joint infections, and medical device-associated bloodstream infections, depend upon its capacity to form tenacious biofilms on surfaces. Inserted medical devices such as intravenous catheters, pacemakers, and artificial heart valves save lives, but unfortunately, they can also serve as a substrate on which S. aureus can form a biofilm, attributing S. aureus as a leading cause of medical device-related infections. The major aim of this work was take compounds to which S. aureus would be exposed during infection and to investigate their effects on its capacity to form a biofilm. More specifically, the project investigated the effects of serum, and thereafter of catheter lock solutions on biofilm formation by S. aureus. Pre-coating polystyrene with serum is frequently used as a method to augment biofilm formation. The effect of pre-coating with serum is due to the deposition of extracellular matrix components onto the polystyrene, which are then recognized by MSCRAMMs. We therefore hypothesized that the major component of blood, serum, would induce biofilm formation. Surprisingly, serum actually inhibited biofilm formation. The inhibitory activity was due to a small molecular weight, heat-stable, non-proteinaceous component/s of serum. Serum-mediated inhibition of biofilm formation may represent a previously uncharacterized aspect of host innate immunity that targets the expression of a key bacterial virulence factor: the ability to establish a resistant biofilm. Metal ion chelators like sodium citrate are frequently chosen to lock intravenous catheters because they are regarded as potent inhibitors of bacterial biofilm formation and viability. We found that, while chelating compounds abolished biofilm formation in most strains of S. aureus, they actually augmented the phenotype in a subset of strains. We

  9. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

    International Nuclear Information System (INIS)

    Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng; Shi, Xianglin; Kim, Jong-Ghee; Heo, Jung Sun; Choe, Youngji; Jeon, Young-Mi; Lee, Jeong-Chae

    2012-01-01

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G 2 /M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

  10. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen Ngoc, Tam Dan [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Son, Young-Ok [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Lim, Shin-Saeng [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Kim, Jong-Ghee [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Heo, Jung Sun [Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Choe, Youngji [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Jeon, Young-Mi, E-mail: young@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Lee, Jeong-Chae, E-mail: leejc88@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2012-03-15

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

  11. Effect of GABA agonists and GABA-A receptor modulators on cocaine- and food-maintained responding and cocaine discrimination in rats.

    Science.gov (United States)

    Barrett, Andrew C; Negus, S Stevens; Mello, Nancy K; Caine, S Barak

    2005-11-01

    Recent studies indicate that GABAergic ligands modulate abuse-related effects of cocaine. The goal of this study was to evaluate the effects of a mechanistically diverse group of GABAergic ligands on the discriminative stimulus and reinforcing effects of cocaine in rats. One group of rats was trained to discriminate 5.6 mg/kg cocaine from saline in a two-lever, food-reinforced, drug discrimination procedure. In two other groups, responding was maintained by cocaine (0-3.2 mg/kg/injection) or liquid food (0-100%) under a fixed ratio 5 schedule. Six GABA agonists were tested: the GABA-A receptor agonist muscimol, the GABA-B receptor agonist baclofen, the GABA transaminase inhibitor gamma-vinyl-GABA (GVG), and three GABA-A receptor modulators (the barbiturate pentobarbital, the high-efficacy benzodiazepine midazolam, and the low-efficacy benzodiazepine enazenil). When tested alone, none of the compounds substituted fully for the discriminative stimulus effects of cocaine. As acute pretreatments, select doses of midazolam and pentobarbital produced 2.2- to 3.6-fold rightward shifts in the cocaine dose-effect function. In contrast, muscimol, baclofen, GVG, and enazenil failed to alter the discriminative stimulus effects of cocaine. In assays of cocaine- and food-maintained responding, midazolam and pentobarbital decreased cocaine self-administration at doses 9.6- and 3.3-fold lower, respectively, than those that decreased food-maintained responding. In contrast, muscimol, baclofen, and GVG decreased cocaine self-administration at doses that also decreased food-maintained responding. Enazenil failed to alter cocaine self-administration. Together with previous studies, these data suggest that among mechanistically diverse GABA agonists, high-efficacy GABA-A modulators may be the most effective for modifying the abuse-related effects of cocaine.

  12. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    International Nuclear Information System (INIS)

    Xu, Yuan; Cardell, Lars-Olaf

    2014-01-01

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B 2 receptor agonist) and des-Arg 9 -bradykinin- (selective B 1 receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE 2 . The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg 9 -bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B 2 receptors, but not those on B 1 . Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in some patients with asthma

  13. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yuan, E-mail: yuan.xu@ki.se; Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  14. Lipoplex size determines lipofection efficiency with or without serum.

    Science.gov (United States)

    Almofti, Mohamad Radwan; Harashima, Hideyoshi; Shinohara, Yasuo; Almofti, Ammar; Li, Wenhao; Kiwada, Hiroshi

    2003-01-01

    In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.

  15. β-Arrestin-2-Dependent Signaling Promotes CCR4-mediated Chemotaxis of Murine T-Helper Type 2 Cells.

    Science.gov (United States)

    Lin, Rui; Choi, Yeon Ho; Zidar, David A; Walker, Julia K L

    2018-06-01

    Allergic asthma is a complex inflammatory disease that leads to significant healthcare costs and reduction in quality of life. Although many cell types are implicated in the pathogenesis of asthma, CD4 + T-helper cell type 2 (Th2) cells are centrally involved. We previously reported that the asthma phenotype is virtually absent in ovalbumin-sensitized and -challenged mice that lack global expression of β-arrestin (β-arr)-2 and that CD4 + T cells from these mice displayed significantly reduced CCL22-mediated chemotaxis. Because CCL22-mediated activation of CCR4 plays a role in Th2 cell regulation in asthmatic inflammation, we hypothesized that CCR4-mediated migration of CD4 + Th2 cells to the lung in asthma may use β-arr-dependent signaling. To test this hypothesis, we assessed the effect of various signaling inhibitors on CCL22-induced chemotaxis using in vitro-polarized primary CD4 + Th2 cells from β-arr2-knockout and wild-type mice. Our results show, for the first time, that CCL22-induced, CCR4-mediated Th2 cell chemotaxis is dependent, in part, on a β-arr2-dependent signaling pathway. In addition, we show that this chemotactic signaling mechanism involves activation of P-p38 and Rho-associated protein kinase. These findings point to a proinflammatory role for β-arr2-dependent signaling and support β-arr2 as a novel therapeutic target in asthma.

  16. Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ruochan [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Fu, Sha; Fan, Xue-Gong [Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Lotze, Michael T.; Zeh, Herbert J. [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Tang, Daolin, E-mail: tangd2@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Kang, Rui, E-mail: kangr@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2015-03-13

    High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis and necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.

  17. Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner

    DEFF Research Database (Denmark)

    Kny, Melanie; Standera, Sybille; Hartmann-Petersen, Rasmus

    2011-01-01

    in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We...

  18. Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

    Science.gov (United States)

    Collins, Denis M; Gately, Kathy; Hughes, Clare; Edwards, Connla; Davies, Anthony; Madden, Stephen F; O'Byrne, Kenneth J; O'Donovan, Norma; Crown, John

    2017-09-01

    Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high

  19. Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.

    Science.gov (United States)

    Sumegi, Mate; Fukazawa, Yugo; Matsui, Ko; Lorincz, Andrea; Eyre, Mark D; Nusser, Zoltan; Shigemoto, Ryuichi

    2012-04-01

    Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)Rγ2(77I)lox) in which the γ2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (γ2(77I)) and used viral vectors to swap γ2(77I) with wild-type, zolpidem-sensitive γ2 subunits (γ2(77F)). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABA(A)Rγ2(−/−) mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 ((AU1)γ2(77F)) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the (AU1)γ2(77F) subunit (Cre-2A-(AU1)γ2(77F)) into the neocortex of GABA(A)Rγ2(77I)lox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the (AU1)γ2(77F) subunits in perisomatic GABAergic synapses. In line with this,miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ2(77I) and (AU1)γ2(77F) GABA(A)Rs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it

  20. Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

    International Nuclear Information System (INIS)

    O'Connell, A.R.; Lee, B.W.; Stenson-Cox, C.

    2006-01-01

    Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events

  1. Pentylenetetrazol modulates redox system by inducing addicsin translocation from endoplasmic reticulum to plasma membrane in NG108-15 cells

    Directory of Open Access Journals (Sweden)

    Mitsushi J. Ikemoto

    2017-09-01

    Full Text Available Addicsin (Arl6ip5 is a multifunctional physiological and pathophysiological regulator that exerts its effects by readily forming homo- and hetero-complexes with various functional factors. In particular, addicsin acts as a negative modulator of neural glutamate transporter excitatory amino acid carrier 1 (EAAC1 and participates in the regulation of intracellular glutathione (GSH content by negatively modulating EAAC1-mediated cysteine and glutamate uptake. Addicsin is considered to play a crucial role in the onset of neurodegenerative diseases including epilepsy. However, the molecular dynamics of addicsin remains largely unknown. Here, we report the dynamics of addicsin in NG108-15 cells upon exposure to pentylenetetrazol (PTZ, a representative epileptogenic agent acting on the gamma-Aminobutyric acid A (GABAA receptor. Fluorescent immunostaining analysis demonstrated that addicsin drastically changed its localization from the endoplasmic reticulum (ER to the plasma membrane within 1 h of PTZ exposure in a dose-dependent manner. Moreover, addicsin was co-localized with the plasma membrane markers EAAC1 and Na+/K+ ATPase alpha-3 upon PTZ stimulation. This translocation was significantly inhibited by a non-competitive GABAA receptor antagonist, picrotoxin, but not by a competitive GABAA receptor antagonist, bicuculline. Furthermore, lactate dehydrogenase (LDH assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH radical-scavenging assay showed that PTZ-induced addicsin translocation was accompanied by a decrease of radical-scavenging activity and an increase of cytotoxicity in a PTZ dose-dependent manner. These findings suggest that PTZ induces the translocation of addicsin from the ER to the plasma membrane and modulates the redox system by regulating EAAC1-mediated GSH synthesis, which leads to the activation of cell death signaling.

  2. NMDA-dependent phase synchronization between septal and temporal CA3 hippocampal networks.

    Science.gov (United States)

    Gu, Ning; Jackson, Jesse; Goutagny, Romain; Lowe, Germaine; Manseau, Frédéric; Williams, Sylvain

    2013-05-08

    Increasing evidence suggests that synchronization between brain regions is essential for information exchange and memory processes. However, it remains incompletely known which synaptic mechanisms contribute to the process of synchronization. Here, we investigated whether NMDA receptor-mediated synaptic plasticity was an important player in synchronization between septal and temporal CA3 areas of the rat hippocampus. We found that both the septal and temporal CA3 regions intrinsically generate weakly synchronized δ frequency oscillations in the complete hippocampus in vitro. Septal and temporal oscillators differed in frequency, power, and rhythmicity, but both required GABAA and AMPA receptors. NMDA receptor activation, and most particularly the NR2B subunit, contributed considerably more to rhythm generation at the temporal than the septal region. Brief activation of NMDA receptors by application of extracellular calcium dramatically potentiated the septal-temporal coherence for long durations (>40 min), an effect blocked by the NMDA antagonist AP-5. This long-lasting NMDA-receptor-dependent increase in coherence was also associated with an elevated phase locking of spikes locally and across regions. Changes in coherence between oscillators were associated with increases in phase locking between oscillators independent of oscillator amplitude. Finally, although the septal CA3 rhythm preceded the oscillations in temporal regions in control conditions, this was reversed during the NMDA-dependent enhancement in coherence, suggesting that NMDA receptor activation can change the direction of information flow along the septotemporal CA3 axis. These data demonstrate that plastic changes in communication between septal and temporal hippocampal regions can arise from the NMDA-dependent phase locking of neural oscillators.

  3. Gamma-aminobutyric acid (GABA) stimulates pancreatic cancer growth through overexpressing GABAA receptor pi subunit.

    Science.gov (United States)

    Takehara, Akio; Hosokawa, Masayo; Eguchi, Hidetoshi; Ohigashi, Hiroaki; Ishikawa, Osamu; Nakamura, Yusuke; Nakagawa, Hidewaki

    2007-10-15

    Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter in the mature central nervous system, and GABA/GABA receptors are also present in nonneural tissues, including cancer, but their precise function in nonneuronal or cancerous cells has thus far been poorly defined. Through the genome-wide cDNA microarray analysis of pancreatic ductal adenocarcinoma (PDAC) cells as well as subsequent reverse transcription-PCR and Northern blot analyses, we identified the overexpression of GABA receptor pi subunit (GABRP) in PDAC cells. We also found the expression of this peripheral type GABAA receptor subunit in few adult human organs. Knockdown of endogenous GABRP expression in PDAC cells by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in PDAC cell viability. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRP-expressing PDAC cells, but not GABRP-negative cells, and GABAA receptor antagonists inhibited this growth-promoting effect by GABA. The HEK293 cells constitutively expressing exogenous GABRP revealed the growth-promoting effect of GABA treatment. Furthermore, GABA treatment in GABRP-positive cells increased intracellular Ca2+ levels and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) cascade. Clinical PDAC tissues contained a higher level of GABA than normal pancreas tissues due to the up-regulation of glutamate decarboxylase 1 expression, suggesting their autocrine/paracrine growth-promoting effect in PDACs. These findings imply that GABA and GABRP could play important roles in PDAC development and progression, and that this pathway can be a promising molecular target for the development of new therapeutic strategies for PDAC.

  4. Functional characterization of GABAA receptor-mediated modulation of cortical neuron network activity in microelectrode array recordings

    DEFF Research Database (Denmark)

    Bader, Benjamin M; Steder, Anne; Klein, Anders Bue

    2017-01-01

    The numerous γ-aminobutyric acid type A receptor (GABAAR) subtypes are differentially expressed and mediate distinct functions at neuronal level. In this study we have investigated GABAAR-mediated modulation of the spontaneous activity patterns of primary neuronal networks from murine frontal...... of the information extractable from the MEA recordings offers interesting insights into the contributions of various GABAAR subtypes/subgroups to cortical network activity and the putative functional interplay between these receptors in these neurons....... cortex by characterizing the effects induced by a wide selection of pharmacological tools at a plethora of activity parameters in microelectrode array (MEA) recordings. The basic characteristics of the primary cortical neurons used in the recordings were studied in some detail, and the expression levels...

  5. Effect of serum proteins on polystyrene nanoparticle uptake and intracellular trafficking in endothelial cells

    International Nuclear Information System (INIS)

    Guarnieri, Daniela; Guaccio, Angela; Fusco, Sabato; Netti, Paolo A.

    2011-01-01

    The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge and surface functionalization, control their capability to interact with cells and, in particular, with sub-cellular components. This interaction can be also influenced by the adsorption of molecules present in biological fluids, like blood, on NP surface. Here, we analysed the effect of serum proteins on 49 and 100 nm red fluorescent polystyrene NP uptake in porcine aortic endothelial (PAE) cells, as a model for vascular transport. To this aim, NP uptake kinetic, endocytic pathway and intracellular trafficking were studied by monitoring NPs inside cells through confocal microscopy and multiple particle tracking (MPT). We demonstrated that NPs are rapidly internalized by cells in serum-free (SF) medium, according to a saturation kinetic. Conversely, in 10% foetal bovine serum-enriched (SE) medium, NP uptake rate results drastically reduced. Moreover, NP internalization depends on an active endocytic mechanism that does not involve clathrin- and caveolae-mediated vesicular transport, in both SE and SF media. Furthermore, MPT data indicate that NP intracellular trafficking is unaffected by protein presence. Indeed, approximately 50–60% of internalized NPs is characterized by a sub-diffusive behaviour, whereas the remaining fraction shows an active motion. These findings demonstrate that the unspecific protein adsorption on NP surface can affect cellular uptake in terms of internalization kinetics, but it is not effective in controlling active and cellular-mediated uptake mechanisms of NPs and their intracellular routes.

  6. Treatment of serum with supernatants from cultures of Candida albicans reduces its serum-dependent phagocytosis Tratamento de soro com sobrenadante de cultura de Candida albicans reduz a fagocitose soro-dependente

    Directory of Open Access Journals (Sweden)

    Aderbal Antonio dos Santos

    2002-01-01

    Full Text Available Candida albicans is a potent activator of the complement system, and heat labile opsonins produced by activation of C3 (C3b and iC3b enhance phagocytosis of C. albicans mediated by complement receptors. In this study we treated mouse serum with supernatants from cultures of a protease producer strain of C. albicans and evaluated the ability of this serum to enhance phagocytosis of C. albicans. Cell-free supernatants from cultures of C. albicans were concentrated 5 fold and added to mouse serum for 30 min at 37ºC, before using this serum for opsonization of glutaraldehyde-fixed yeast cells. We observed that normal mouse serum increased about 3 fold the phagocytosis of C. albicans by mice peritoneal macrophages, whereas supernatant-treated serum did not increase phagocytosis. This effect of supernatants on serum was prevented by addition of pepstatin (5 µg/ ml; an inhibitor of C. albicans acid proteases to the medium. Serum treated with supernatants from cultures of a protease-deficient mutant of C. albicans also increased about 3 fold phagocytosis of the yeast. These results suggest that a protease produced by C. albicans causes proteolysis of serum opsonins, thereby reducing the phagocytosis of the yeast.Candida albicans é um potente ativador do sistema complemento, e opsoninas lábeis ao calor produzidas por ativação de C3 (C3b e iC3b aumentam a fagocitose de C. albicans mediada por receptores de complemento. Neste estudo, tratamos o soro de camundongo com sobrenadante de culturas de uma cepa de C. albicans produtora de proteases e avaliamos a capacidade deste soro reduzir a fagocitose de C. albicans. Sobrenadantes livres de células obtidos de cultura de C. albicans foram concentrados 5 vezes e adicionados ao soro de camundongo por 30 minutos a 37ºC, antes deste soro ser usado para opsonização de C. albicans na forma de levedura e fixadas em glutaraldeido. Nós observamos que soro normal aumentou 3 vezes a fagocitose de C. albicans por

  7. Dynamic volume changes in astrocytes are an intrinsic phenomenon mediated by bicarbonate ion flux.

    Directory of Open Access Journals (Sweden)

    Clare M Florence

    Full Text Available Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in extracellular potassium ([K(+](o and pH homeostasis. Pathological brain states that result in [K(+](o and pH dysregulation have been shown to cause astrocyte swelling. However, whether astrocyte volume changes occur under physiological conditions is not known. In this study we used two-photon imaging to visualize real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were observed to swell by 19.0±0.9% in response to a small physiological increase in the concentration of [K(+](o (3 mM. Astrocyte swelling was mediated by the influx of bicarbonate (HCO(3- ions as swelling was significantly decreased when the influx of HCO(3- was reduced. We found: 1 in HCO(3- free extracellular solution astrocytes swelled by 5.4±0.7%, 2 when the activity of the sodium-bicarbonate cotransporter (NBC was blocked the astrocytes swelled by 8.3±0.7%, and 3 in the presence of an extracellular carbonic anhydrase (CA inhibitor astrocytes swelled by 11.4±0.6%. Because a significant HCO(3- efflux is known to occur through the γ-amino-butyric acid (GABA channel, we performed a series of experiments to determine if astrocytes were capable of HCO(3- mediated volume shrinkage with GABA channel activation. Astrocytes were found to shrink -7.7±0.5% of control in response to the GABA(A channel agonist muscimol. Astrocyte shrinkage from GABA(A channel activation was significantly decreased to -5.0±0.6% of control in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ. These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.

  8. The role of heat shock protein 70 in mediating age-dependent mortality in sepsis.

    Science.gov (United States)

    McConnell, Kevin W; Fox, Amy C; Clark, Andrew T; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A; Farris, Alton B; Buchman, Timothy G; Hunt, Clayton R; Coopersmith, Craig M

    2011-03-15

    Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6- to 12-wk-old) and aged (16- to 17-mo-old) HSP70(-/-) and wild-type (WT) mice to determine whether HSP70 modulated outcome in an age-dependent fashion. Young HSP70(-/-) and WT mice subjected to cecal ligation and puncture, Pseudomonas aeruginosa pneumonia, or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70(-/-) mice than aged WT mice subjected to cecal ligation and puncture (p = 0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared with WT mice, aged septic HSP70(-/-) mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70(-/-) mice had increased systemic levels of TNF-α, IL-6, IL-10, and IL-1β compared with WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged, but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation.

  9. Melanopsin mediates light-dependent relaxation in blood vessels.

    Science.gov (United States)

    Sikka, Gautam; Hussmann, G Patrick; Pandey, Deepesh; Cao, Suyi; Hori, Daijiro; Park, Jong Taek; Steppan, Jochen; Kim, Jae Hyung; Barodka, Viachaslau; Myers, Allen C; Santhanam, Lakshmi; Nyhan, Daniel; Halushka, Marc K; Koehler, Raymond C; Snyder, Solomon H; Shimoda, Larissa A; Berkowitz, Dan E

    2014-12-16

    Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4(-/-) mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.

  10. Scavenger receptor-mediated endocytosis by sinusoidal cells in rat bone marrow

    International Nuclear Information System (INIS)

    Geoffroy, J.S.

    1987-01-01

    Endocytosis of serum albumin by sinusoidal endothelial cells in rat bone marrow was investigated initially at the ultrastructural level with subsequent biochemical investigation of the specificity mediating this event. Bovine serum albumin adsorbed to 20nm colloidal gold particles (AuBSA) was chosen as the electron microscopic probe. Morphological data strongly suggested that a receptor was involved in uptake of AuBSA. Confirmation of receptor involvement in the uptake of AuBSA by marrow sinusoidal endothelial cells was achieved utilizing an in situ isolated hind limb perfusion protocol in conjunction with unlabeled, radiolabeled, and radio-/colloidal gold labeled probes. The major findings of competition and saturation experiments were: (1) endocytosis of AuBSA was mediated by a receptor for modified/treated serum albumin; (2) endocytosis of formaldehyde-treated serum albumin was mediated by a binding site which may be the same or closely related to the site responsible for the uptake of AuBSA; and (3) endocytosis of native untreated albumin was not mediated by receptor and probably represents fluid-phase pinocitosis

  11. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor gamma2 subunit.

    Science.gov (United States)

    Kittler, Josef T; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R; McAinsh, Kristina; Arancibia-Carcamo, I Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J

    2008-03-04

    The regulation of the number of gamma2-subunit-containing GABA(A) receptors (GABA(A)Rs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface gamma2-subunit-containing GABA(A)Rs is regulated. Here, we identify a gamma2-subunit-specific Yxxvarphi-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for gamma2-subunit tyrosine phosphorylation. Blocking GABA(A)R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxvarphi motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that gamma2-subunit-containing heteromeric GABA(A)Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA(A)R surface levels and synaptic inhibition.

  12. Identification and cloning of a gamma 3 subunit splice variant of the human GABA(A) receptor.

    Science.gov (United States)

    Poulsen, C F; Christjansen, K N; Hastrup, S; Hartvig, L

    2000-05-31

    cDNA sequences encoding two forms of the GABA(A) gamma 3 receptor subunit were cloned from human hippocampus. The nucleotide sequences differ by the absence (gamma 3S) or presence (gamma 3L) of 18 bp located in the presumed intracellular loop between transmembrane region (TM) III and IV. The extra 18 bp in the gamma 3L subunit generates a consensus site for phosphorylation by protein kinase C (PKC). Analysis of human genomic DNA encoding the gamma 3 subunit reveals that the 18 bp insert is contiguous with the upstream proximal exon.

  13. Experience-Dependent Equilibration of AMPAR-Mediated Synaptic Transmission during the Critical Period

    Directory of Open Access Journals (Sweden)

    Kyung-Seok Han

    2017-01-01

    Full Text Available Experience-dependent synapse refinement is essential for functional optimization of neural circuits. However, how sensory experience sculpts excitatory synaptic transmission is poorly understood. Here, we show that despite substantial remodeling of synaptic connectivity, AMPAR-mediated synaptic transmission remains at equilibrium during the critical period in the mouse primary visual cortex. The maintenance of this equilibrium requires neurogranin (Ng, a postsynaptic calmodulin-binding protein important for synaptic plasticity. With normal visual experience, loss of Ng decreased AMPAR-positive synapse numbers, prevented AMPAR-silent synapse maturation, and increased spine elimination. Importantly, visual deprivation halted synapse loss caused by loss of Ng, revealing that Ng coordinates experience-dependent AMPAR-silent synapse conversion to AMPAR-active synapses and synapse elimination. Loss of Ng also led to sensitized long-term synaptic depression (LTD and impaired visually guided behavior. Our synaptic interrogation reveals that experience-dependent coordination of AMPAR-silent synapse conversion and synapse elimination hinges upon Ng-dependent mechanisms for constructive synaptic refinement during the critical period.

  14. Direct and indirect associations between social anxiety and nicotine dependence and cessation problems: multiple mediator analyses.

    Science.gov (United States)

    Buckner, Julia D; Farris, Samantha G; Schmidt, Norman B; Zvolensky, Michael J

    2014-06-01

    Little empirical work has evaluated why socially anxious smokers are especially vulnerable to more severe nicotine dependence and cessation failure. Presumably, these smokers rely on cigarettes to help them manage their chronically elevated negative affect elicited by a wide array of social contexts. The current study examined the direct and indirect effects of social anxiety cross-sectionally in regard to a range of smoking processes among 466 treatment-seeking smokers. Negative affect and negative affect reduction motives were examined as mediators of the relations of social anxiety with nicotine dependence and cessation problems. Social anxiety was directly and robustly associated with perceived barriers to smoking cessation and problems experienced during past quit attempts. Social anxiety was also associated with greater nicotine dependence and smoking inflexibility indirectly through negative affect and negative affect smoking motives. Negative affect and smoking to reduce negative affect mediated these relations. These findings document the important role of negative affect and negative affect reduction motives in the relationships of social anxiety with nicotine dependence and cessation problems.

  15. Valence and atomic size dependent exchange barriers in vacancy-mediated dopant diffusion

    International Nuclear Information System (INIS)

    Nelson, J.S.; Schultz, P.A.; Wright, A.F.

    1998-01-01

    First-principles pseudopotential calculations of dopant-vacancy exchange barriers indicate a strong dependency on dopant valence and atomic size, in contrast to current models of vacancy-mediated dopant diffusion. First-row elements (B, C, N) are found to have exchange barriers which are an order of magnitude larger than the assumed value of 0.3 eV (the Si vacancy migration energy). copyright 1998 American Institute of Physics

  16. Interactions of L-3,5,3'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes

    Science.gov (United States)

    Westergard, Thomas; Salari, Reza; Martin, Joseph V.; Brannigan, Grace

    2015-01-01

    Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3’-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone. PMID:26421724

  17. Investigation on the pH-dependent binding of Eosin Y and bovine serum albumin by spectral methods

    International Nuclear Information System (INIS)

    Gao Dejiang; Tian Yuan; Liang Fanghui; Jin Danhong; Chen Yanhua; Zhang Hanqi; Yu Aimin

    2007-01-01

    In this paper, the pH-dependent binding of Eosin Y and bovine serum albumin (BSA) was investigated by spectral methods, including resonance light scattering (RLS), absorption and fluorescence spectrometry. Due to the pH-dependent structure of Eosin Y and BSA, the interaction of BSA and Eosin Y depended on the solution pH value. Especially at pH 2.6 and 9.2, the RLS intensity of BSA was obviously enhanced in the presence of Eosin Y. However, the fluorescence intensity of BSA was quenched in the presence of Eosin Y. To fully understand the pH-dependent binding of BSA and Eosin Y, fluorescence quenching technique was introduced. Based on the fluorescence data obtained, the style of binding, the binding constant, the binding site number and the thermodynamic parameters for the interaction of BSA and Eosin Y were studied. Based on Foerster non-radiation energy transfer theory, the distance between donor BSA and acceptor Eosin Y was obtained

  18. Investigation on the pH-dependent binding of Eosin Y and bovine serum albumin by spectral methods

    Energy Technology Data Exchange (ETDEWEB)

    Gao Dejiang; Tian Yuan [College of Chemistry, Jilin University, Changchun 130012 (China); Liang Fanghui; Jin Danhong [Changchun Medical College, Changchun 130031 (China); Chen Yanhua; Zhang Hanqi [College of Chemistry, Jilin University, Changchun 130012 (China); Yu Aimin [College of Chemistry, Jilin University, Changchun 130012 (China)], E-mail: analchem@mail.jlu.edu.cn

    2007-12-15

    In this paper, the pH-dependent binding of Eosin Y and bovine serum albumin (BSA) was investigated by spectral methods, including resonance light scattering (RLS), absorption and fluorescence spectrometry. Due to the pH-dependent structure of Eosin Y and BSA, the interaction of BSA and Eosin Y depended on the solution pH value. Especially at pH 2.6 and 9.2, the RLS intensity of BSA was obviously enhanced in the presence of Eosin Y. However, the fluorescence intensity of BSA was quenched in the presence of Eosin Y. To fully understand the pH-dependent binding of BSA and Eosin Y, fluorescence quenching technique was introduced. Based on the fluorescence data obtained, the style of binding, the binding constant, the binding site number and the thermodynamic parameters for the interaction of BSA and Eosin Y were studied. Based on Foerster non-radiation energy transfer theory, the distance between donor BSA and acceptor Eosin Y was obtained.

  19. GM-CSF production from human airway smooth muscle cells is potentiated by human serum

    Directory of Open Access Journals (Sweden)

    Maria B. Sukkar

    2000-01-01

    Full Text Available Recent evidence suggests that airway smooth muscle cells (ASMC actively participate in the airway inflammatory process in asthma. Interleukin–1β (IL–1β and tumour necrosis factor–α (TNF–α induce ASMC to release inflammatory mediators in vitro. ASMC mediator release in vivo, however, may be influenced by features of the allergic asthmatic phenotype. We determined whether; (1 allergic asthmatic serum (AAS modulates ASMC mediator release in response to IL–1β and TNF–α, and (2 IL–1β/TNF–α prime ASMC to release mediators in response to AAS. IL–5 and GMCSF were quantified by ELISA in culture supernatants of; (1 ASMC pre-incubated with either AAS, non-allergic non-asthmatic serum (NAS or MonomedTM (a serum substitute and subsequently stimulated with IL–1β and TNF–α and (2 ASMC stimulated with IL–1β/TNF–α and subsequently exposed to either AAS, NAS or MonomedTM. IL-1g and TNF–α induced GM-CSF release in ASMC pre-incubated with AAS was not greater than that in ASMC pre-incubated with NAS or MonomedTM. IL–1β and TNF–α, however, primed ASMC to release GM-CSF in response to human serum. GM-CSF production following IL–1β/TNF–α and serum exposure (AAS or NAS was significantly greater than that following IL–1β /TNF–α and MonomedTM exposure or IL–1β/TNF–α exposure only. Whilst the potentiating effects of human serum were not specific to allergic asthma, these findings suggest that the secretory capacity of ASMC may be up-regulated during exacerbations of asthma, where there is evidence of vascular leakage.

  20. Toxicity of isoproturon on Saccharomyces cerevisiae growing in mineral medium depends on glutathione-mediated antioxidant capacity.

    OpenAIRE

    Candeias, M; Alves-Pereira, I; Ferreira, Rui

    2011-01-01

    The results revealed an increase of viable cells, after 72 h of culture and an increase of antioxidant power mediated by GSH and GR activity in S. cerevisiae UE-ME3. The adaptive response of UE-ME3 strain to isoproturon, determined in MB, was clearly higher than observed in IGC-3507 strain. So, we presume that the extent of the toxic effect of isoproturon in both yeast strains depends on glutathione-mediated antioxidant capacity.

  1. Effect of hydromorphone hydrochloride combined with ropivacaine for PCEA after orthopedic surgery on the synthesis of pain mediators, inflammatory mediator and oxygen free radicals

    Directory of Open Access Journals (Sweden)

    Liang-Ying Luo

    2017-08-01

    Full Text Available Objective: To explore the effect of hydromorphone hydrochloride combined with ropivacaine for PCEA after orthopedic surgery on the synthesis of pain mediators, inflammatory mediator and oxygen free radicals. Methods: A total of 120 patients with fracture who underwent operation in the hospital between July 2014 and December 2016 were collected and divided into control group and observation group according to the random number table method, 60 cases in each group. Control group received morphine hydrochloride combined with ropivacaine for analgesia, observation group received hydromorphone hydrochloride combined with ropivacaine for analgesia, and the postoperative analgesia lasted for 48 h. The differences in serum levels of pain mediators, inflammatory mediators and oxidative stress indexes were compared between the two groups. Results: Immediately after operation, the differences in serum levels of pain mediators, inflammatory mediators and oxidative stress indexes were not statistically significant between the two groups. 48 h after operation, serum PGE2, SP, β-EP, IL-6, MCP-1, HMGB-1 and MDA levels of both groups of patients were significantly lower than those immediately after operation while Cu-Zn SOD and GSH-Px levels were significantly higher than those immediately after operation, and serum PGE2, SP, β-EP, IL-6, MCP-1, HMGB-1 and MDA levels of observation group were significantly lower than those of control group while Cu-Zn SOD and GSH-Px levels were significantly higher than those of control group. Conclusion: Hydromorphone hydrochloride combined with ropivacaine for PCEA after orthopedic surgery is effective in alleviating pain and inhibiting systemic inflammatory response.

  2. Hypoxia-induced hypothermia mediated by GABA in the rostral parapyramidal area of the medulla oblongata.

    Science.gov (United States)

    Osaka, T

    2014-05-16

    Hypoxia evokes a regulated decrease in the body core temperature (Tc) in a variety of animals. The neuronal mechanisms of this response include, at least in part, glutamatergic activation in the lateral preoptic area (LPO) of the hypothalamus. As the sympathetic premotor neurons in the medulla oblongata constitute a cardinal relay station in the descending neuronal pathway from the hypothalamus for thermoregulation, their inhibition can also be critically involved in the mechanisms of the hypoxia-induced hypothermia. Here, I examined the hypothesis that hypoxia-induced hypothermia is mediated by glutamate-responsive neurons in the LPO that activate GABAergic transmission in the rostral raphe pallidus (rRPa) and neighboring parapyramidal region (PPy) of the medulla oblongata in urethane-chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. Unilateral microinjection of GABA (15nmol) into the rRPa and PPy regions elicited a prompt increase in tail skin temperature (Ts) and decreases in Tc, oxygen consumption rate (VO2), and heart rate. Next, when the GABAA receptor blocker bicuculline methiodide (bicuculline methiodide (BMI), 10pmol) alone was microinjected into the rRPa, it elicited unexpected contradictory responses: simultaneous increases in Ts, VO2 and heart rate and a decrease in Tc. Then, when BMI was microinjected bilaterally into the PPy, no direct effect on Ts was seen; and thermogenic and tachycardic responses were slight. However, pretreatment of the PPy with BMI, but not vehicle saline, greatly attenuated the hypothermic responses evoked by hypoxic (10%O2-90%N2, 5min) ventilation or bilateral microinjections of glutamate (5nmol, each side) into the LPO. The results suggest that hypoxia-induced hypothermia was mediated, at least in part, by the activation of GABAA receptors in the PPy. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Cr(VI) induces mitochondrial-mediated and caspase-dependent apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41 cells

    International Nuclear Information System (INIS)

    Son, Young-Ok; Hitron, J. Andrew; Wang Xin; Chang Qingshan; Pan Jingju; Zhang Zhuo; Liu Jiankang; Wang Shuxia; Lee, Jeong-Chae; Shi Xianglin

    2010-01-01

    Cr(VI) compounds are known to cause serious toxic and carcinogenic effects. Cr(VI) exposure can lead to a severe damage to the skin, but the mechanisms involved in the Cr(VI)-mediated toxicity in the skin are unclear. The present study examined whether Cr(VI) induces cell death by apoptosis or necrosis using mouse skin epidermal cell line, JB6 Cl41 cells. We also investigated the cellular mechanisms of Cr(VI)-induced cell death. This study showed that Cr(VI) induced apoptotic cell death in a dose-dependent manner, as demonstrated by the appearance of cell shrinkage, the migration of cells into the sub-G1 phase, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cr(VI) treatment resulted in the increases of mitochondrial membrane depolarization and caspases activation. Electron spin resonance (ESR) and fluorescence analysis revealed that Cr(VI) increased intracellular levels of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion radical in dose-dependent manner. Blockage of p53 by si-RNA transfection suppressed mitochondrial changes of Bcl-2 family composition, mitochondrial membrane depolarization, caspase activation and PARP cleavage, leading to the inhibition of Cr(VI)-induced apoptosis. Further, catalase treatment prevented p53 phosphorylation stimulated by Cr(VI) with the concomitant inhibition of caspase activation. These results suggest that Cr(VI) induced a mitochondrial-mediated and caspase-dependent apoptosis in skin epidermal cells through activation of p53, which are mainly mediated by reactive oxidants generated by the chemical.

  4. Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6

    DEFF Research Database (Denmark)

    Mathiesen, Christian K; Jensen, Tanja B; Prentoe, Jannick

    2014-01-01

    Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1-6 serum-free HCVcc (sf-HCVcc) from Huh7.......5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6-2.1 log10 higher infectivity titers (4.7-6.2 log10 Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single......-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture...

  5. Grb2 mediates semaphorin-4D-dependent RhoA inactivation.

    Science.gov (United States)

    Sun, Tianliang; Krishnan, Rameshkumar; Swiercz, Jakub M

    2012-08-01

    Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.

  6. K-Cl Cotransporter 2–mediated Cl− Extrusion Determines Developmental Stage–dependent Impact of Propofol Anesthesia on Dendritic Spines

    KAUST Repository

    Puskarjov, Martin; Fiumelli, Hubert; Briner, Adrian; Bodogan, Timea; Demeter, Kornel; Lacoh, Claudia Marvine; Mavrovic, Martina; Blaesse, Peter; Kaila, Kai; Vutskits, Laszlo

    2017-01-01

    Background: General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABA A)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). Methods: In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABA A)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. Results: The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl - transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. Conclusions: The KCC2-dependent developmental increase in the efficacy of GABA A -mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

  7. K-Cl Cotransporter 2–mediated Cl− Extrusion Determines Developmental Stage–dependent Impact of Propofol Anesthesia on Dendritic Spines

    KAUST Repository

    Puskarjov, Martin

    2017-03-16

    Background: General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABA A)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). Methods: In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABA A)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. Results: The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl - transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. Conclusions: The KCC2-dependent developmental increase in the efficacy of GABA A -mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

  8. Functional modifications of acid-sensing ion channels by ligand-gated chloride channels.

    Directory of Open Access Journals (Sweden)

    Xuanmao Chen

    Full Text Available Together, acid-sensing ion channels (ASICs and epithelial sodium channels (ENaC constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR. Here we show that ASICs were reversibly inhibited by activation of GABA(A receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A receptor-mediated currents. Moreover, activation of the GABA(A receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A receptors, also modified ASICs in spinal neurons. We conclude that GABA(A receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.

  9. Parahippocampal Cortex Mediates the Relationship between Lutein and Crystallized Intelligence in Healthy, Older Adults.

    Science.gov (United States)

    Zamroziewicz, Marta K; Paul, Erick J; Zwilling, Chris E; Johnson, Elizabeth J; Kuchan, Matthew J; Cohen, Neal J; Barbey, Aron K

    2016-01-01

    Introduction: Although, diet has a substantial influence on the aging brain, the relationship between dietary nutrients and aspects of brain health remains unclear. This study examines the neural mechanisms that mediate the relationship between a carotenoid important for brain health across the lifespan, lutein, and crystallized intelligence in cognitively intact older adults. We hypothesized that higher serum levels of lutein are associated with better performance on a task of crystallized intelligence, and that this relationship is mediated by gray matter structure of regions within the temporal cortex. This investigation aims to contribute to a growing line of evidence, which suggests that particular nutrients may slow or prevent aspects of cognitive decline by targeting specific features of brain aging. Methods: We examined 76 cognitively intact adults between the ages of 65 and 75 to investigate the relationship between serum lutein, tests of crystallized intelligence (measured by the Wechsler Abbreviated Scale of Intelligence), and gray matter volume of regions within the temporal cortex. A three-step mediation analysis was implemented using multivariate linear regressions to control for age, sex, education, income, depression status, and body mass index. Results: The mediation analysis revealed that gray matter thickness of one region within the temporal cortex, the right parahippocampal cortex (Brodmann's Area 34), partially mediates the relationship between serum lutein and crystallized intelligence. Conclusion: These results suggest that the parahippocampal cortex acts as a mediator of the relationship between serum lutein and crystallized intelligence in cognitively intact older adults. Prior findings substantiate the individual relationships reported within the mediation, specifically the links between (i) serum lutein and temporal cortex structure, (ii) serum lutein and crystallized intelligence, and (iii) parahippocampal cortex structure and

  10. p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.

    Science.gov (United States)

    Lafarga, Vanesa; Cuadrado, Ana; Nebreda, Angel R

    2007-10-01

    Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38alpha (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38alpha as well as the activation of specific target genes by p53. We recently characterized a new p38alpha substrate, named p18(Hamlet) (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18(Hamlet) protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that p18(Hamlet) can also mediate the cell cycle arrest induced in response to gamma-irradiation, by participating in the p53-dependent upregulation of the cell cycle inhibitor p21(Cip1) (CDKN1A).

  11. Menadione induces the formation of reactive oxygen species and depletion of GSH-mediated apoptosis and inhibits the FAK-mediated cell invasion.

    Science.gov (United States)

    Kim, Yun Jeong; Shin, Yong Kyoo; Sohn, Dong Suep; Lee, Chung Soo

    2014-09-01

    Menadione induces apoptosis in tumor cells. However, the mechanism of apoptosis in ovarian cancer cells exposed to menadione is not clear. In addition, it is unclear whether menadione-induced apoptosis is mediated by the depletion of glutathione (GSH) contents that is associated with the formation of reactive oxygen species. Furthermore, the effect of menadione on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effects of menadione exposure on apoptosis, cell adhesion, and cell migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that menadione may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of menadione appears to be mediated by the formation of reactive oxygen species and the depletion of GSH. Menadione inhibited fetal-bovine-serum-induced cell adhesion and migration of OVCAR-3 cells, possibly through the suppression the focal adhesion kinase (FAK)-dependent activation of cytoskeletal-associated components. Therefore, menadione might be beneficial in the treatment of epithelial ovarian adenocarcinoma and combination therapy.

  12. Interactions of L-3,5,3'-Triiodothyronine [corrected], Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

    Science.gov (United States)

    Westergard, Thomas; Salari, Reza; Martin, Joseph V; Brannigan, Grace

    2015-01-01

    Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

  13. How microelectrode array-based chick forebrain neuron biosensors respond to glutamate NMDA receptor antagonist AP5 and GABAA receptor antagonist musimol

    Directory of Open Access Journals (Sweden)

    Serena Y. Kuang

    2016-09-01

    Full Text Available We have established a long-term, stable primary chick forebrain neuron (FBN culture on a microelectrode array platform as a biosensor system for neurotoxicant screening and for neuroelectrophysiological studies for multiple purposes. This paper reports some of our results, which characterize the biosensor pharmacologically. Dose-response experiments were conducted using NMDA receptor antagonist AP5 and GABAA receptor agonist musimol (MUS. The chick FBN biosensor (C-FBN-biosensor responds to the two agents in a pattern similar to that of rodent counterparts; the estimated EC50s (the effective concentration that causes 50% inhibition of the maximal effect are 2.3 μM and 0.25 μM, respectively. Intercultural and intracultural reproducibility and long-term reusability of the C-FBN-biosensor are addressed and discussed. A phenomenon of sensitization of the biosensor that accompanies intracultural reproducibility in paired dose-response experiments for the same agent (AP5 or MUS is reported. The potential application of the C-FBN-biosensor as an alternative to rodent biosensors in shared sensing domains (NMDA receptor and GABAA receptor is suggested. Keywords: Biosensor, Microelectrode array, Neurotoxicity, Chick forebrain neuron, AP5, Musimol

  14. Serum level of soluble fibrinogen-like protein 2 in renal allograft recipients with acute rejection: a preliminary study.

    Science.gov (United States)

    Zhao, Z; Yang, C; Tang, Q; Zhao, T; Jia, Y; Ma, Z; Rong, R; Xu, M; Zhu, T

    2012-12-01

    Soluble fibrinogen-like protein 2 (sfgl2), which is mainly secreted by T cells, is a novel effector of regulatory T cells with immunosuppressive functions. The aim of this study was to investigate serum levels of sfgl2 among renal allograft recipients. From November 2010 to August 2011 we retrospectively divided 47 renal allograft recipients into an acute rejection (n = 19) versus a stable group (n = 28) according to allograft biopsy results, using the Banff 2007 classification. The acute rejection group was subdivided into grade I (n = 8) versus grade II T-cell-mediated (n = 6) or antibody-mediated rejection episodes (n = 5). Peripheral blood samples were collected at the time of biopsy. Fourteen healthy volunteers were included as normal group controls. Serum levels of sfgl2 were analyzed by enzyme-linked immunosorbent assay. Serum levels of sfgl2 were increased among renal allograft recipients suffering from biopsy-proven acute rejection episodes (61.91 ± 45.68 ng/mL), versus those with stable allografts (38.59 ± 19.92 ng/mL, P rejection episodes (41.71 ± 16.44 ng/mL, P rejection (34.10 ± 9.26 ng/mL, P rejection episodes to an extent dependent upon the pathological type and severity of the response. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  15. Serum Levels of the Adipokine Progranulin Depend on Renal Function

    Science.gov (United States)

    Richter, Judit; Focke, Denise; Ebert, Thomas; Kovacs, Peter; Bachmann, Anette; Lössner, Ulrike; Kralisch, Susan; Kratzsch, Jürgen; Beige, Joachim; Anders, Matthias; Bast, Ingolf; Blüher, Matthias; Stumvoll, Michael; Fasshauer, Mathias

    2013-01-01

    OBJECTIVE Progranulin has recently been introduced as a novel adipokine inducing insulin resistance and obesity. In the current study, we investigated renal elimination, as well as association of the adipokine with markers of the metabolic syndrome. RESEARCH DESIGN AND METHODS Progranulin serum levels were quantified by enzyme-linked immunosorbent assay and correlated to anthropometric and biochemical parameters of renal function and glucose and lipid metabolism, as well as inflammation, in 532 patients with stages 1–5 of chronic kidney disease (CKD). RESULTS Median serum progranulin levels adjusted for age, sex, and BMI were significantly different between CKD stages with highest values detectable in stage 5 (stage 1, 58.3 µg/L; stage 2, 63.0 µg/L; stage 3, 65.4 µg/L; stage 4, 68.8 µg/L; and stage 5, 90.6 µg/L). Furthermore, CKD stage was the strongest independent predictor of circulating progranulin in our cohort. In addition, high-sensitivity interleukin-6 and adiponectin remained significantly and independently correlated with the adipokine. CONCLUSIONS We demonstrate that progranulin serum levels increase with deteriorating renal function. These findings are in accordance with the hypothesis that renal clearance is a major elimination route for circulating progranulin. Furthermore, the adipokine is positively and independently associated with markers of inflammation and adiponectin. PMID:23033238

  16. Roles of thioredoxin in nitric oxide-dependent preconditioning-induced tolerance against MPTP neurotoxin

    International Nuclear Information System (INIS)

    Chiueh, C.C.; Andoh, Tsugunobu; Chock, P. Boon

    2005-01-01

    Hormesis, a stress tolerance, can be induced by ischemic preconditioning stress. In addition to preconditioning, it may be induced by other means, such as gas anesthetics. Preconditioning mechanisms, which may be mediated by reprogramming survival genes and proteins, are obscure. A known neurotoxicant, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes less neurotoxicity in the mice that are preconditioned. Pharmacological evidences suggest that the signaling pathway of ·NO-cGMP-PKG (protein kinase G) may mediate preconditioning phenomenon. We developed a human SH-SY5Y cell model for investigating · NO-mediated signaling pathway, gene regulation, and protein expression following a sublethal preconditioning stress caused by a brief 2-h serum deprivation. Preconditioned human SH-SY5Y cells are more resistant against severe oxidative stress and apoptosis caused by lethal serum deprivation and 1-mehtyl-4-phenylpyridinium (MPP + ). Both sublethal and lethal oxidative stress caused by serum withdrawal increased neuronal nitric oxide synthase (nNOS/NOS1) expression and · NO levels to a similar extent. In addition to free radical scavengers, inhibition of nNOS, guanylyl cyclase, and PKG blocks hormesis induced by preconditioning. S-nitrosothiols and 6-Br-cGMP produce a cytoprotection mimicking the action of preconditioning tolerance. There are two distinct cGMP-mediated survival pathways: (i) the up-regulation of a redox protein thioredoxin (Trx) for elevating mitochondrial levels of antioxidant protein Mn superoxide dismutase (MnSOD) and antiapoptotic protein Bcl-2, and (ii) the activation of mitochondrial ATP-sensitive potassium channels [K(ATP)]. Preconditioning induction of Trx increased tolerance against MPP + , which was blocked by Trx mRNA antisense oligonucleotide and Trx reductase inhibitor. It is concluded that Trx plays a pivotal role in · NO-dependent preconditioning hormesis against MPTP/MPP +

  17. Tyrosine isomers mediate the classical phenomenon of concomitant tumor resistance.

    Science.gov (United States)

    Ruggiero, Raúl A; Bruzzo, Juan; Chiarella, Paula; di Gianni, Pedro; Isturiz, Martín A; Linskens, Susana; Speziale, Norma; Meiss, Roberto P; Bustuoabad, Oscar D; Pasqualini, Christiane D

    2011-11-15

    Concomitant tumor resistance (CR) is a phenomenon originally described in 1906 in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although recent studies have indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In this study, we identify this serum factor as tyrosine in its meta and ortho isoforms. In three different murine models of cancer that generate CR, both meta-tyrosine and ortho-tyrosine inhibited tumor growth. In addition, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isoforms were mediated, in part, by early inhibition of mitogen-activated protein/extracellular signal-regulated kinase pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors, an issue of pivotal importance to oncologists and their patients. ©2011 AACR

  18. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A receptor-mediated signaling.

    Directory of Open Access Journals (Sweden)

    Anna Kakehashi

    Full Text Available Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA A receptor (GABA(AR system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(AR agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN. Formation of glutathione S-transferase placental form positive (GST-P(+ foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(AR alpha 1 subunit was observed in GST-P(+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+ foci by activating GABA(AR-mediated signaling.

  19. Interleukin-10 overexpression promotes Fas-ligand-dependent chronic macrophage-mediated demyelinating polyneuropathy.

    Directory of Open Access Journals (Sweden)

    Dru S Dace

    Full Text Available BACKGROUND: Demyelinating polyneuropathy is a debilitating, poorly understood disease that can exist in acute (Guillain-Barré syndrome or chronic forms. Interleukin-10 (IL-10, although traditionally considered an anti-inflammatory cytokine, has also been implicated in promoting abnormal angiogenesis in the eye and in the pathobiology of autoimmune diseases such as lupus and encephalomyelitis. PRINCIPAL FINDINGS: Overexpression of IL-10 in a transgenic mouse model leads to macrophage-mediated demyelinating polyneuropathy. IL-10 upregulates ICAM-1 within neural tissues, promoting massive macrophage influx, inflammation-induced demyelination, and subsequent loss of neural tissue resulting in muscle weakness and paralysis. The primary insult is to perineural myelin followed by secondary axonal loss. Infiltrating macrophages within the peripheral nerves demonstrate a highly pro-inflammatory signature. Macrophages are central players in the pathophysiology, as in vivo depletion of macrophages using clodronate liposomes reverses the phenotype, including progressive nerve loss and paralysis. Macrophage-mediate demyelination is dependent on Fas-ligand (FasL-mediated Schwann cell death. SIGNIFICANCE: These findings mimic the human disease chronic idiopathic demyelinating polyneuropathy (CIDP and may also promote further understanding of the pathobiology of related conditions such as acute idiopathic demyelinating polyneuropathy (AIDP or Guillain-Barré syndrome.

  20. Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABAA receptor clustering induced by inhibitory synaptic plasticity

    KAUST Repository

    Orlando, Marta

    2017-10-17

    Both excitatory and inhibitory synaptic contacts display activity dependent dynamic changes in their efficacy that are globally termed synaptic plasticity. Although the molecular mechanisms underlying glutamatergic synaptic plasticity have been extensively investigated and described, those responsible for inhibitory synaptic plasticity are only beginning to be unveiled. In this framework, the ultrastructural changes of the inhibitory synapses during plasticity have been poorly investigated. Here we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) to characterize the fine structural rearrangements of post-synaptic GABAA Receptors (GABAARs) at the nanometric scale during the induction of inhibitory long-term potentiation (iLTP). Additional electron tomography (ET) experiments on immunolabelled hippocampal neurons allowed the visualization of synaptic contacts and confirmed the reorganization of post-synaptic GABAAR clusters in response to chemical iLTP inducing protocol. Altogether, these approaches revealed that, following the induction of inhibitory synaptic potentiation, GABAAR clusters increase in size and number at the post-synaptic membrane with no other major structural changes of the pre- and post-synaptic elements.

  1. Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABAA receptor clustering induced by inhibitory synaptic plasticity

    KAUST Repository

    Orlando, Marta; Ravasenga, Tiziana; Petrini, Enrica Maria; Falqui, Andrea; Marotta, Roberto; Barberis, Andrea

    2017-01-01

    Both excitatory and inhibitory synaptic contacts display activity dependent dynamic changes in their efficacy that are globally termed synaptic plasticity. Although the molecular mechanisms underlying glutamatergic synaptic plasticity have been extensively investigated and described, those responsible for inhibitory synaptic plasticity are only beginning to be unveiled. In this framework, the ultrastructural changes of the inhibitory synapses during plasticity have been poorly investigated. Here we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) to characterize the fine structural rearrangements of post-synaptic GABAA Receptors (GABAARs) at the nanometric scale during the induction of inhibitory long-term potentiation (iLTP). Additional electron tomography (ET) experiments on immunolabelled hippocampal neurons allowed the visualization of synaptic contacts and confirmed the reorganization of post-synaptic GABAAR clusters in response to chemical iLTP inducing protocol. Altogether, these approaches revealed that, following the induction of inhibitory synaptic potentiation, GABAAR clusters increase in size and number at the post-synaptic membrane with no other major structural changes of the pre- and post-synaptic elements.

  2. Coping mediates the effects of cognitive-behavioral therapy for alcohol use disorder among out-patient clients in Project MATCH when dependence severity is high.

    Science.gov (United States)

    Roos, Corey R; Maisto, Stephen A; Witkiewitz, Katie

    2017-09-01

    There is inconsistent evidence that alcohol-specific coping is a mechanism of change in cognitive-behavioral therapy (CBT) for alcohol use disorder (AUD). Our primary aim was to test whether baseline dependence severity moderates the mediational effect of CBT on drinking outcomes via coping. Secondary data analysis of Project MATCH , a multi-site alcohol treatment trial in which participants, recruited in out-patient and aftercare arms, were randomized to three treatments: CBT, motivational enhancement therapy (MET) and Twelve-Step facilitation (TSF). Nine research sites in the United States. A total of 1063 adults with AUD. The primary outcomes were percentage days abstinent and percentage heavy drinking days at the 1-year follow-up. Coping was assessed with the Processes of Change Questionnaire . Dependence severity was measured with the Alcohol Dependence Scale . Among the full available sample (across treatment arms), there were no significant moderated mediation effects. Double moderated mediation analyses indicated that several moderated mediation effects were moderated by treatment arm (all P cognitive-behavioral therapy for alcohol use disorder was conditional on dependence severity. End-of-treatment coping mediated the positive treatment effects of cognitive-behavioral therapy on 1-year drinking outcomes among out-patient clients when dependence severity was high, but not when dependence severity was low or moderate. © 2017 Society for the Study of Addiction.

  3. Exploring the Mediational Role of Coping Motives for Marijuana Use in Terms of the Relation between Anxiety Sensitivity and Marijuana Dependence

    OpenAIRE

    Johnson, Kirsten; Mullin, Jennifer L.; Marshall, Erin C.; Bonn-Miller, Marcel O.; Zvolensky, Michael

    2010-01-01

    The current study evaluated the prediction that coping motives for marijuana use would mediate the relation between anxiety sensitivity and a marijuana dependence diagnosis after controlling for other co-occurring marijuana use motives. Participants were 136 current marijuana users (47.1% women; Mage = 21.9, SD = 7.2). Results were consistent with a mediational effect, with the relation between anxiety sensitivity and marijuana dependence being explained by the addition of coping motives into...

  4. Zinc in human serum

    International Nuclear Information System (INIS)

    Kiilerich, S.

    1987-01-01

    The zinc ion is essential for the living organism. Many pathological conditions have been described as a consequence of zinc deficiency. As zinc constitutes less than 0.01 per cent of the body weight, it conventionally belongs to the group of trace elements. The method of atomic absorption spectrophotometry is used to measure the concentration of zinc in serum and urine from healthy persons. The assumptions of the method is discussed. The importance of proteinbinding, diet and the diurnal variation of serum zinc concentration is presented. Serum versus plasma zinc concentration is discussed. Reference serum zinc values from 104 normal subjects are given. Zinc in serum is almost entirely bound to proteins. A preliminary model for the estimation of the distribution of zinc between serum albumin and α 2 -macroglobulin is set up. This estimate has been examined by an ultracentrufugation method. The binding of zinc to a α 2 -macroglobulin in normal persons is appoximately 7 per cent, in patients with cirrhosis of the liver of alcoholic origin approximately 6 per cent, in patients with insulin dependent diabetes mellitus approximately 5 per cent, and in patients with chronic renal failure approximately 2 per cent. It is concluded, therefore, that for clinical purposes it is sufficient to use the concentration of total serum zinc corrected for the concentration of serum albumin. (author)

  5. Functional analysis of Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Hein, Estrid; Honoré, Christian; Skjoedt, Mikkel-Ole

    2010-01-01

    Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation...... was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides...

  6. Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent.

    Directory of Open Access Journals (Sweden)

    Carmen M Dickinson-Copeland

    Full Text Available Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10 in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB damage and mortality. Endothelial progenitor cells (EPC are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC's are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02 and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.

  7. Parahippocampal Cortex Mediates the Relationship between Lutein and Crystallized Intelligence in Healthy, Older Adults

    Directory of Open Access Journals (Sweden)

    Marta Karolina Zamroziewicz

    2016-12-01

    Full Text Available Introduction: Although diet has a substantial influence on the aging brain, the relationship between dietary nutrients and aspects of brain health remains unclear. This study examines the neural mechanisms that mediate the relationship between a carotenoid important for brain health across the lifespan, lutein, and crystallized intelligence in cognitively intact older adults. We hypothesized that higher serum levels of lutein are associated with better performance on a task of crystallized intelligence, and that this relationship is mediated by gray matter structure of regions within the temporal cortex. This investigation aims to contribute to a growing line of evidence, which suggests that particular nutrients may slow or prevent aspects of cognitive decline by targeting specific features of brain aging.Methods: We examined 75 cognitively intact adults between the ages of 65 and 75 to investigate the relationship between serum lutein, tests of crystallized intelligence (measured by the Wechsler Abbreviated Scale of Intelligence, and gray matter volume of regions within the temporal cortex. A three-step mediation analysis was implemented using multivariate linear regressions to control for age, sex, education, income, depression status, and body mass index.Results: The mediation analysis revealed that gray matter thickness of one region within the temporal cortex, the right parahippocampal cortex (Brodmann’s Area 34, partially mediates the relationship between serum lutein and crystallized intelligence. Conclusion: These results suggest that the parahippocampal cortex acts as a mediator of the relationship between serum lutein and crystallized intelligence in cognitively intact older adults. Prior findings substantiate the individual relationships reported within the mediation, specifically the links between (i serum lutein and temporal cortex structure, (ii serum lutein and crystallized intelligence, and (iii parahippocampal cortex structure

  8. Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development

    International Nuclear Information System (INIS)

    Lin ChenTao; Ahmad, M.; Cashmore, A.R.

    1996-01-01

    Cryptochrome 1 (CRY1) is a flavin-type blue type receptor of Arabidopsis thaliana which mediates inhibition of hypocotyl elongation. In the work described in this report it is demonstrated that CRY1 is a soluble protein expressed in both young seedlings grown either in the dark or under light, and in different organs of adult plants. The functional role of CRY1 was further investigated using transgenic Arabidopsis plants overexpressing CRY1. It is demonstrated that overexpression of CRY1 resulted in hypersensitivity to blue, UV-A, and green light for the inhibition of hypocotyl elongation response. Transgenic plants overexpressing CRY1 also exhibited a dwarf phenotype with reduced size in almost every organ. This was in keeping with the previous observation of reciprocal alterations found in hy4 mutant plants and is consistent with a hypothesis that CRY1 mediates a light-dependent process resulting in a general inhibitory effect on plant growth. In addition, transgenic plants overexpressing CRY1 showed increased anthocyanin accumulation in response to blue, UV-A, and green light in a fluence rate-dependent manner. This increase in anthocyanin accumulation in transgenic plants was shown to be concomitant with increased blue light-induction of CHS gene expression. It is concluded that CRY1 is a photoreceptor mediating blue light-dependent regulation of gene expression in addition to its affect on plant growth. (author)

  9. Human serum albumin mediated self-assembly of gold nanoparticles into hollow spheres

    Energy Technology Data Exchange (ETDEWEB)

    Nayak, Nimai C [Singapore-MIT Alliance, Manufacturing Systems and Technology Programme, Nanyang Technological University, 65 Nanyang Drive, 637460 (Singapore); Shin, Kwanwoo [Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsoo-dong, Mapo-gu, Seoul 121-742 (Korea, Republic of)], E-mail: ncnayak@gmail.com

    2008-07-02

    The assembly of nanoparticles in topologically predefined superstructures is an important area in nanoscale architecture. In this paper, we report an unusual aggregation phenomenon involving L-lysine capped gold nanoparticles and human serum albumin into hollow nanospheres. The electrostatic interaction between positively charged L-lysine capped gold nanoparticles and negatively charged human serum albumin at physiological pH led to the assembly of the gold nanoparticles into hollow spheres. The phenomenon can be explained by the dry hole opening mechanism.

  10. Human serum albumin mediated self-assembly of gold nanoparticles into hollow spheres

    International Nuclear Information System (INIS)

    Nayak, Nimai C; Shin, Kwanwoo

    2008-01-01

    The assembly of nanoparticles in topologically predefined superstructures is an important area in nanoscale architecture. In this paper, we report an unusual aggregation phenomenon involving L-lysine capped gold nanoparticles and human serum albumin into hollow nanospheres. The electrostatic interaction between positively charged L-lysine capped gold nanoparticles and negatively charged human serum albumin at physiological pH led to the assembly of the gold nanoparticles into hollow spheres. The phenomenon can be explained by the dry hole opening mechanism

  11. Duplicated Gephyrin Genes Showing Distinct Tissue Distribution and Alternative Splicing Patterns Mediate Molybdenum Cofactor Biosynthesis, Glycine Receptor Clustering, and Escape Behavior in Zebrafish*

    Science.gov (United States)

    Ogino, Kazutoyo; Ramsden, Sarah L.; Keib, Natalie; Schwarz, Günter; Harvey, Robert J.; Hirata, Hiromi

    2011-01-01

    Gephyrin mediates the postsynaptic clustering of glycine receptors (GlyRs) and GABAA receptors at inhibitory synapses and molybdenum-dependent enzyme (molybdoenzyme) activity in non-neuronal tissues. Gephyrin knock-out mice show a phenotype resembling both defective glycinergic transmission and molybdenum cofactor (Moco) deficiency and die within 1 day of birth due to starvation and dyspnea resulting from deficits in motor and respiratory networks, respectively. To address whether gephyrin function is conserved among vertebrates and whether gephyrin deficiency affects molybdoenzyme activity and motor development, we cloned and characterized zebrafish gephyrin genes. We report here that zebrafish have two gephyrin genes, gphna and gphnb. The former is expressed in all tissues and has both C3 and C4 cassette exons, and the latter is expressed predominantly in the brain and spinal cord and harbors only C4 cassette exons. We confirmed that all of the gphna and gphnb splicing isoforms have Moco synthetic activity. Antisense morpholino knockdown of either gphna or gphnb alone did not disturb synaptic clusters of GlyRs in the spinal cord and did not affect touch-evoked escape behaviors. However, on knockdown of both gphna and gphnb, embryos showed impairments in GlyR clustering in the spinal cord and, as a consequence, demonstrated touch-evoked startle response behavior by contracting antagonistic muscles simultaneously, instead of displaying early coiling and late swimming behaviors, which are executed by side-to-side muscle contractions. These data indicate that duplicated gephyrin genes mediate Moco biosynthesis and control postsynaptic clustering of GlyRs, thereby mediating key escape behaviors in zebrafish. PMID:20843816

  12. Dynamic changes in GABAA receptors on basal forebrain cholinergic neurons following sleep deprivation and recovery

    Directory of Open Access Journals (Sweden)

    Jones Barbara E

    2007-02-01

    Full Text Available Abstract Background The basal forebrain (BF cholinergic neurons play an important role in cortical activation and arousal and are active in association with cortical activation of waking and inactive in association with cortical slow wave activity of sleep. In view of findings that GABAA receptors (Rs and inhibitory transmission undergo dynamic changes as a function of prior activity, we investigated whether the GABAARs on cholinergic cells might undergo such changes as a function of their prior activity during waking vs. sleep. Results In the brains of rats under sleep control (SC, sleep deprivation (SD or sleep recovery (SR conditions in the 3 hours prior to sacrifice, we examined immunofluorescent staining for β2–3 subunit GABAARs on choline acetyltransferase (ChAT immunopositive (+ cells in the magnocellular BF. In sections also stained for c-Fos, β2–3 GABAARs were present on ChAT+ neurons which expressed c-Fos in the SD group alone and were variable or undetectable on other ChAT+ cells across groups. In dual-immunostained sections, the luminance of β2–3 GABAARs over the membrane of ChAT+ cells was found to vary significantly across conditions and to be significantly higher in SD than SC or SR groups. Conclusion We conclude that membrane GABAARs increase on cholinergic cells as a result of activity during sustained waking and reciprocally decrease as a result of inactivity during sleep. These changes in membrane GABAARs would be associated with increased GABA-mediated inhibition of cholinergic cells following prolonged waking and diminished inhibition following sleep and could thus reflect a homeostatic process regulating cholinergic cell activity and thereby indirectly cortical activity across the sleep-waking cycle.

  13. The mediating role of self-criticism and dependency in the association between perceptions of maternal caring and depressive symptoms.

    Science.gov (United States)

    Campos, Rui C; Besser, Avi; Blatt, Sidney J

    2010-12-01

    This study examined a theoretically based mediation model including participants' perceptions of early relationships with their mother, self-criticism, dependency, and current depressive symptoms. We expect that (a) early relationships characterized by low levels of care and high levels of overprotection will be positively associated with both current depressive state and self-criticism and dependency; (b) high levels of self-criticism and dependency will be positively associated with depressive symptoms; and (c) self-criticism and dependency will play a mediating role in the association between participants' perceptions of early relationships characterized by low levels of care and high levels of overprotection and their current depressive symptoms. A nonclinical community sample of 200 Portuguese adults participated in the study. Perceptions of early relationships were measured using the mother scales of the Parental Bonding Instrument (Parker et al. [1979: Br J Med Psychol 52:1-10]), levels of self-criticism and dependency were measured using the Depressive Experiences Questionnaire (Blatt et al. [1976: J Abn Psy 6:383-389]), and depressive symptoms were measured using the Center for the Epidemiological Studies of Depression Scale (Radloff [1977: Appl Psychol Meas 1:385-401]. Structural equation modeling showed that the link between participants' perceptions of early caretaking relationships with their mothers and their current depressive symptoms is mediated by high levels of self-criticism--a personality trait associated with vulnerability to depression--but not Dependency. However, an ancillary analysis indicated that the link between participants' perceptions of early maternal overprotective relationships and their current depressive symptoms is mediated by high levels of Neediness. Findings underscore the role of perceived early relationships in psychological vulnerability to depression among highly self-critical and among highly needy individuals and

  14. An NMDA Receptor-Dependent Mechanism Underlies Inhibitory Synapse Development

    Directory of Open Access Journals (Sweden)

    Xinglong Gu

    2016-01-01

    Full Text Available In the mammalian brain, GABAergic synaptic transmission provides inhibitory balance to glutamatergic excitatory drive and controls neuronal output. The molecular mechanisms underlying the development of GABAergic synapses remain largely unclear. Here, we report that NMDA-type ionotropic glutamate receptors (NMDARs in individual immature neurons are the upstream signaling molecules essential for GABAergic synapse development, which requires signaling via Calmodulin binding motif in the C0 domain of the NMDAR GluN1 subunit. Interestingly, in neurons lacking NMDARs, whereas GABAergic synaptic transmission is strongly reduced, the tonic inhibition mediated by extrasynaptic GABAA receptors is increased, suggesting a compensatory mechanism for the lack of synaptic inhibition. These results demonstrate a crucial role for NMDARs in specifying the development of inhibitory synapses, and suggest an important mechanism for controlling the establishment of the balance between synaptic excitation and inhibition in the developing brain.

  15. Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

    Science.gov (United States)

    Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

    2015-02-01

    Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. The role of Omi/HtrA2 protease in neonatal postasphyxial serum-induced apoptosis in human kidney proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Zhang Yong

    2012-01-01

    Full Text Available Omi/HtrA2, a proapoptotic mitochondrial serine protease, is involved in both caspase-dependent and caspaseindependent apoptosis. A growing body of evidence indicates that Omi/HtrA2 plays an important role in the pathogenesis of a variety of ischemia-reperfusion (I/R injuries. However, the role of Omi/HtrA2 in renal injuries that occur in neonates with asphyxia remains unknown. The present study was designed to investigate whether Omi/HtrA2 plays an important role in the types of renal injuries that are induced by neonatal postasphyxial serum. Human renal proximal tubular cell line (HK-2 cells were used as targets. A 20% serum taken from neonates one day after asphyxia was applied to target cells as an attacking factor. We initially included control and postasphyxial serum-attacked groups and later included a ucf-101 group in the study. In the postasphyxial serum-treated group, cytosolic Omi/HtrA2 and caspase-3 expression in HK-2 cells was significantly higher than in the control group. Moreover, the concentration of cytosolic caspase-3 was found to be markedly decreased in HK-2 cells in the ucf-101 group. Our results suggest both that postasphyxial serum has a potent apoptosis-inducing effect on HK-2 cells and that this effect can be partially blocked by ucf-101. Taken together, our results demonstrate for the first time that postasphyxial serum from neonates results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it promotes HK-2 cell apoptosis via a protease activity-dependent, caspase-mediated pathway.

  17. TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins.

    Science.gov (United States)

    Tomlinson, Gillian; Chimalapati, Suneeta; Pollard, Tracey; Lapp, Thabo; Cohen, Jonathan; Camberlein, Emilie; Stafford, Sian; Periselneris, Jimstan; Aldridge, Christine; Vollmer, Waldemar; Picard, Capucine; Casanova, Jean-Laurent; Noursadeghi, Mahdad; Brown, Jeremy

    2014-10-01

    Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae. Copyright © 2014 The Authors.

  18. Intravenous infusion of docosahexaenoic acid increases serum concentrations in a dose-dependent manner and increases seizure latency in the maximal PTZ model.

    Science.gov (United States)

    Trépanier, Marc-Olivier; Kwong, Kei-Man; Domenichiello, Anthony F; Chen, Chuck T; Bazinet, Richard P; Burnham, W M

    2015-09-01

    Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (n-3 PUFA) that has been shown to raise seizure thresholds in the maximal pentylenetetrazole model following acute subcutaneous (s.c.) administration in rats. Following s.c. administration, however, the dose-response relationship for DHA has shown an inverted U-pattern. The purposes of the present experiment were as follows: (1) to determine the pattern of serum unesterified concentrations resulting from the intravenous (i.v.) infusions of various doses of DHA, (2) to determine the time course of these concentrations following the discontinuation of the infusions, and (3) to determine whether seizure protection in the maximal PTZ model would correlate with serum unesterified DHA levels. Animals received 5-minute i.v. infusions of saline or 25, 50, 100, or 200mg/kg of DHA via a cannula inserted into one of the tail veins. Blood was collected during and after the infusions by means of a second cannula inserted into the other tail vein (Experiment 1). A separate group of animals received saline or 12.5-, 25-, 50-, 100-, or 200 mg/kg DHA i.v. via a cannula inserted into one of the tail veins and were then seizure-tested in the maximal PTZ model either during infusion or after the discontinuation of the infusions. Slow infusions of DHA increased serum unesterified DHA concentrations in a dose-dependent manner, with the 200-mg/kg dose increasing the concentration approximately 260-fold compared with saline-infused animals. Following discontinuation of the infusions, serum concentrations rapidly dropped toward baseline, with half-lives of approximately 40 and 11s for the 25-mg/kg dose and 100-mg/kg dose, respectively. In the seizure-tested animals, DHA significantly increased latency to seizure onset in a dose-dependent manner. Following the discontinuation of infusion, seizure latency rapidly decreased toward baseline. Overall, our study suggests that i.v. infusion of unesterified DHA results in

  19. Novel time-dependent vascular actions of Δ9-tetrahydrocannabinol mediated by peroxisome proliferator-activated receptor gamma

    International Nuclear Information System (INIS)

    O'Sullivan, Saoirse E.; Tarling, Elizabeth J.; Bennett, Andrew J.; Kendall, David A.; Randall, Michael D.

    2005-01-01

    Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, Δ 9 -tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPARγ). In vitro, THC (10 μM) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPARγ agonist rosiglitazone and was inhibited by the PPARγ antagonist GW9662 (1 μM), but not the cannabinoid CB 1 receptor antagonist AM251 (1 μM). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPARγ, transiently expressed in combination with retinoid X receptor α and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 μM). In vitro incubation with THC (1 or 10 μM, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPARγ ligands. The present results provide strong evidence that THC is a PPARγ ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors

  20. The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

    International Nuclear Information System (INIS)

    Geppert, Mark; Petters, Charlotte; Thiel, Karsten; Dringen, Ralf

    2013-01-01

    Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood–brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 ± 2 to 107 ± 6 nm and the zeta-potential of the particles from −22 ± 5 to −9 ± 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

  1. Upregulation of endothelin ETB receptor-mediated vasoconstriction in rat coronary artery after organ culture

    DEFF Research Database (Denmark)

    Eskesen, Karen; Edvinsson, Lars

    2006-01-01

    The aim of this study was to examine if endothelin ET(B) receptor-mediated contraction occurred in isolated segments of rat coronary arteries during organ culture. Presence of contractile endothelin ET(B) receptors was studied by measuring the change in isometric tension in rings of left anterior......(+)-solution was not modified after 1 day in culture medium. The experiments indicate that organ culture of rat coronary arteries upregulate endothelin ET(B) receptor-mediated contraction by inducing synthesis of new protein....... descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET(B) receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction...

  2. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  3. Effects of therapeutic plasma exchange on serum immunoglobulin concentrations in a dog with refractory immune-mediated hemolytic anemia.

    Science.gov (United States)

    Scagnelli, Alyssa M; Walton, Stuart A; Liu, Chin-Chi; Acierno, Mark J

    2018-05-01

    CASE DESCRIPTION A 9-year-old 8.3-kg (18.3-lb) neutered male Miniature Schnauzer was referred for diagnosis and treatment of a sudden onset of lethargy, anorexia, vomiting, and pallor. CLINICAL FINDINGS On physical examination, the dog was lethargic with pale mucous membranes and a capillary refill time ≥ 2 seconds. Skin and sclera were mildly icteric. Signs of pain were elicited during abdominal palpation, and an enlarged spleen was noted. Results of agglutination testing and cytologic findings were consistent with immune-mediated hemolytic anemia (IMHA). No contributing factors for development of IMHA were identified. TREATMENT AND OUTCOME Initial treatment included management with immunosuppressant medications. Three packed RBC transfusions were administered, but clinical signs continued to progress. Therefore, therapeutic plasma exchange (TPE) was performed 5 and 9 days after admission. Following each TPE procedure, the dog had an appreciable clinical improvement and decrease in RBC autoagglutination, and the Hct stabilized. Serum IgG and IgM concentrations were measured during and after both TPE procedures. Despite anticoagulative treatment, the dog developed a thrombus in the splenic vein, necessitating a splenectomy. CLINICAL RELEVANCE The decrease and rebound in serum IgG and IgM concentrations following TPE provided evidence that TPE may have the same immunomodulatory effects in dogs as have been proposed to occur in people. Further, findings suggested that TPE may be a useful alternative in dogs with refractory IMHA when traditional treatments fail.

  4. Uptake of silver nanoparticles by monocytic THP-1 cells depends on particle size and presence of serum proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kettler, Katja, E-mail: K.Kettler@science.ru.nl [Radboud University Nijmegen, Department of Environmental Science (Netherlands); Giannakou, Christina; Jong, Wim H. de [National Institute for Public Health and the Environment (RIVM) (Netherlands); Hendriks, A. Jan [Radboud University Nijmegen, Department of Environmental Science (Netherlands); Krystek, Petra [Philips Innovation Services (Netherlands)

    2016-09-15

    Human health risks by silver nanoparticle (AgNP) exposure are likely to increase due to the increasing number of NP-containing products and demonstrated adverse effects in various cell lines. Unfortunately, results from (toxicity) studies are often based on exposure dose and are often measured only at a fixed time point. NP uptake kinetics and the time-dependent internal cellular concentration are often not considered. Macrophages are the first line of defense against invading foreign agents including NPs. How macrophages deal with the particles is essential for potential toxicity of the NPs. However, there is a considerable lack of uptake studies of particles in the nanometer range and macrophage-like cells. Therefore, uptake rates were determined over 24 h for three different AgNPs sizes (20, 50 and 75 nm) in medium with and without fetal calf serum. Non-toxic concentrations of 10 ng Ag/mL for monocytic THP-1 cells, representing realistic exposure concentration for short-term exposures, were chosen. The uptake of Ag was higher in medium without fetal calf serum and showed increasing uptake for decreasing NP sizes, both on NP mass and on number basis. Internal cellular concentrations reached roughly 32/10 %, 25/18 % and 21/15 % of the nominal concentration in the absence of fetal calf serum/with fetal calf serum for 20-, 50- and 75-nm NPs, respectively. Our research shows that uptake kinetics in macrophages differ for various NP sizes. To increase the understanding of the mechanism of NP toxicity in cells, the process of uptake (timing) should be considered.

  5. Mitochondrial reactive oxygen species regulate the strength of inhibitory GABA-mediated synaptic transmission

    Science.gov (United States)

    Accardi, Michael V.; Daniels, Bryan A.; Brown, Patricia M. G. E.; Fritschy, Jean-Marc; Tyagarajan, Shiva K.; Bowie, Derek

    2014-01-01

    Neuronal communication imposes a heavy metabolic burden in maintaining ionic gradients essential for action potential firing and synaptic signalling. Although cellular metabolism is known to regulate excitatory neurotransmission, it is still unclear whether the brain’s energy supply affects inhibitory signalling. Here we show that mitochondrial-derived reactive oxygen species (mROS) regulate the strength of postsynaptic GABAA receptors at inhibitory synapses of cerebellar stellate cells. Inhibition is strengthened through a mechanism that selectively recruits α3-containing GABAA receptors into synapses with no discernible effect on resident α1-containing receptors. Since mROS promotes the emergence of postsynaptic events with unique kinetic properties, we conclude that newly recruited α3-containing GABAA receptors are activated by neurotransmitter released onto discrete postsynaptic sites. Although traditionally associated with oxidative stress in neurodegenerative disease, our data identify mROS as a putative homeostatic signalling molecule coupling cellular metabolism to the strength of inhibitory transmission.

  6. Mediated and Moderated Effects of Neurocognitive Impairment on Outcomes of Treatment for Substance Dependence and Major Depression

    Science.gov (United States)

    Worley, Matthew J.; Tate, Susan R.; Granholm, Eric; Brown, Sandra A.

    2015-01-01

    Objective Neurocognitive impairment has not consistently predicted substance use treatment outcomes but has been linked to proximal mediators of outcome. These indirect effects have not been examined in adults with substance dependence and co-occurring psychiatric disorders. We examined mediators and moderators of the effects of neurocognitive impairment on substance use among adults in treatment for alcohol or drug dependence and major depression (MDD). Method Participants were veterans (N =197, mean age = 49.3 years, 90% male, 75% Caucasian) in a trial of two group interventions for alcohol/drug dependence and MDD. Measures examined here included intake neurocognitive assessments and percent days drinking (PDD), percent days using drugs (PDDRG), self-efficacy, 12-step affiliation, and depressive symptoms measured every 3 months from intake to the 18-month follow-up. Results Greater intake neurocognitive impairment predicted lower self-efficacy, lower 12-step affiliation, and greater depression severity, and these time-varying variables mediated the effects of impairment on future PDD and PDDRG. The prospective effects of 12-step affiliation on future PDD were greater for those with greater neurocognitive impairment. Impairment also interacted with depression to moderate the effects of 12-step affiliation and self-efficacy on PDD. Adults with greater impairment and currently severe depression had the strongest associations between 12-step affiliation/self-efficacy and future drinking. Conclusions Greater neurocognitive impairment may lead to poorer outcomes from group therapy for alcohol/drug dependence and MDD due to compromised change in therapeutic processes. Distal factors such as neurocognitive impairment can interact with dynamic risk factors to modulate the association between therapeutic processes and future drinking outcomes. PMID:24588403

  7. Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action

    Energy Technology Data Exchange (ETDEWEB)

    Gee, K.W.; Bolger, M.B.; Brinton, R.E.; Coirini, H.; McEwen, B.S.

    1988-08-01

    Further in vitro studies of steroids active at the gamma-aminobutyric acidA (GABAA) receptor regulated Cl- channel labeled by (35S)-t-butylbicyclophosphorothionate ((35S)TBPS) reveal additional structural requirements necessary for activity. Evaluation of selected steroids for activity against TBPS-induced convulsions show similar requirements for activity. Interestingly, steroids (e.g., 5 alpha-pregnan-3 alpha, 20 alpha-diol) were identified that have high potency but limited efficacy as modulators of (35S)TBPS binding. These characteristics are reminiscent of the clinically useful benzodiazepines (BZs) such as clonazepam. However, interactions between the prototypical anesthetic-barbiturate, sodium pentobarbital, and steroids active at the Cl- channel suggest that they do not share a common site of action as allosteric modulators of (35S)TBPS and BZ receptor binding. The most potent steroid evaluated, 5 alpha-pregnan-3 alpha-ol-20-one, modulates (35S)TBPS binding at low concentrations (IC50 approximately 17 nM) in a regionally dependent manner. All (35S)TBPS binding sites appear to be functionally coupled to a steroid modulatory site. Because several of the active steroids are metabolites of progesterone, their ability to inhibit the binding of (3H)promegestrone to the cytosolic progestin receptor in rat uterus was evaluated. Those steroids showing potent activity at the GABAA receptor-Cl- ionophore were inactive at the intracellular progestin receptor. Such specificity coupled with their high potency provide additional support for the hypothesis that some of these steroids may be involved in the homeostatic regulation of brain excitability via the GABAA-BZ receptor complex.

  8. Serum Albumin Levels and Economic Status in Japanese Older Adults.

    Directory of Open Access Journals (Sweden)

    Asami Ota

    Full Text Available Low serum albumin levels are associated with aging and medical conditions such as cancer, liver dysfunction, inflammation, and malnutrition and might be an independent predictor of long-term mortality in healthy older populations. We tested the hypothesis that economic status is associated with serum albumin levels and explained by nutritional and health status in Japanese older adults.We performed a cross-sectional analysis using data from the Japan Gerontological Evaluation study (JAGES. The study participants were 6528 functionally independent residents (3189 men and 3339 women aged ≥65 years living in four municipalities in Aichi prefecture. We used household income as an indicator of economic status. Multiple linear regression was used to compare serum albumin levels in relation to household income, which was classified as low, middle, and high. Additionally, mediation by nutritional and health-related factors was analyzed in multivariable models.With the middle-income group as reference, participants with low incomes had a significantly lower serum albumin level, even after adjustment for sex, age, residential area, education, marital status, and household structure. The estimated mean difference was -0.17 g/L (95% confidence interval, -0.33 to -0.01 g/L. The relation between serum albumin level and low income became statistically insignificant when "body mass index", "consumption of meat or fish", "self-rated health", "presence of medical conditions", "hyperlipidemia", or "respiratory disease "was included in the model.Serum albumin levels were lower in Japanese older adults with low economic status. The decrease in albumin levels appears to be mediated by nutrition and health-related factors with low household incomes. Future studies are needed to reveal the existence of other pathways.

  9. Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment

    International Nuclear Information System (INIS)

    Mota, Linda C.; Barfield, Christina; Hernandez, Juan P.; Baldwin, William S.

    2011-01-01

    Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75 mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating that NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP.

  10. Antinociceptive effects of fisetin against diabetic neuropathic pain in mice: Engagement of antioxidant mechanisms and spinal GABAA receptors.

    Science.gov (United States)

    Zhao, Xin; Li, Xin-Lin; Liu, Xin; Wang, Chuang; Zhou, Dong-Sheng; Ma, Qing; Zhou, Wen-Hua; Hu, Zhen-Yu

    2015-12-01

    Peripheral painful neuropathy is one of the most common complications in diabetes and necessitates improved treatment. Fisetin, a naturally occurring flavonoid, has been reported to exert antidepressant-like effect in previous studies. As antidepressant drugs are employed clinically to treat neuropathic pain, this work aimed to investigate whether fisetin possess beneficial effect on diabetic neuropathic pain and explore the mechanism(s). We subjected mice to diabetes by a single intraperitoneal (i.p.) injection of streptozotocin (200mg/kg), and von Frey test or Hargreaves test was used to assess mechanical allodynia or thermal hyperalgesia, respectively. Chronic treatment of diabetic mice with fisetin not only ameliorated the established symptoms of thermal hyperalgesia and mechanical allodynia, but also arrested the development of neuropathic pain when given at low doses. Although chronic fisetin administration did not impact on the symptom of hyperglycemia in diabetic mice, it reduced exacerbated oxidative stress in tissues of spinal cord, dorsal root ganglion (DRG) and sciatic verve. Furthermore, the analgesic actions of fisetin were abolished by repetitive co-treatment with the reactive oxygen species (ROS) donor tert-butyl hydroperoxide (t-BOOH), but potentiated by the ROS scavenger phenyl-N-tert-butylnitrone (PBN). Finally, acute blockade of spinal GABAA receptors by bicuculline totally counteracted such fisetin analgesia. These findings indicate that chronic fisetin treatment can delay or correct neuropathic hyperalgesia and allodynia in mice with type 1 diabetes. Mechanistically, the present fisetin analgesia may be associated with its antioxidant activity, and spinal GABAA receptors are likely rendered as downstream targets. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. The role of GABAA receptor activity in post-ictal depression period in a rat kindling model of epilepsy

    Directory of Open Access Journals (Sweden)

    Javad Mirnajafi-Zadeh

    2009-02-01

    Full Text Available Introduction: Following the seizure there is an inhibitory period named “post-ictal depressionperiod” which has a depressing effect on the following seizure. In this study, the role of GABAAreceptors on post-ictal depression period was investigated in amygdala kindling model of epilepsy inrat.Materials and Methods: Animals were kindled by electrical stimulation of amygdala. Then, theywere divided into four groups. Different groups of kindled animals were received a second stimulationat 10, 30, 60 and 90 min after the first stimulation and the percentage of suppression of seizureparameters after the second stimulation was calculated. In another four groups, bicucullin, a selectiveGABAA receptor antagonist (100 nM, was intra-cerebroventricularly microinjected 10 min before thesecond stimulation and its effect on the percentage of suppression induced by the first stimulation wasinvestigated. Six animals were used in each group.Results: When the second stimulation was applied at 10 and 30 min after the first stimulation asignificant reduction was observed in the seizure severity and seizure parameters were depressed afterthe second stimulation. Bicucullin microinjection significantly decreased the percentage of depressionin seizure parameters following the second stimulation compared to the animals received the solventalone (P<0.05. This decrease was significant when the second stimulation was applied at 10 and 30min after the first stimulation.Conclusion: Obtained results showed that activity of GABAA receptors by endogenous GABA isone of the mechanisms involved in post-ictal depressing period. However, the blocking of thesereceptors could not completely prevent this period. Thus, other factors have also role in its induction.

  12. Integration of growth factor signals at the c-fos serum response element.

    Science.gov (United States)

    Price, M A; Hill, C; Treisman, R

    1996-04-29

    A transcription factor ternary complex composed of serum response factor (SRF) and a second factor, ternary complex factor (TCF), mediates the response of the c-fos Serum Response Element to growth factors and mitogens. In NIH3T3 fibroblasts, TCF binding is required for transcriptional activation by the SRE in response to activation of the Ras-Raf-ERK pathway. We compared the properties of three members of the TCF family, Elk-1, SAP-1 and SAP-2 (ERP/NET). Although all the proteins contain sequences required for ternary complex formation with SRF, only Elk-1 and SAP-1 appear to interact with the c-fos SRE efficiently in vivo. Each TCF contains a C-terminal activation domain capable of transcriptional activation in response to activation of the Ras-Raf-ERK pathway, and this is dependent on the integrity of S/T-P motifs conserved between all the TCF family members. In contrast, activation of the SRE by whole serum and the mitogenic phospholipid LPA requires SRF binding alone. Constitutively activated members of the Rho subfamily of Ras-like GTPases are also capable of inducing activation of the SRE in the absence of TCF; unlike activated Ras itself, these proteins do not activate the TCFs in NIH3T3 cells. At the SRE, SRF- and TCF-linked signalling pathways act synergistically to potentiate transcription.

  13. Discoidin Domain Receptor 1 Mediates Myosin-Dependent Collagen Contraction

    Directory of Open Access Journals (Sweden)

    Nuno M. Coelho

    2017-02-01

    Full Text Available Discoidin domain receptor 1 (DDR1 is a tyrosine kinase collagen adhesion receptor that mediates cell migration through association with non-muscle myosin IIA (NMIIA. Because DDR1 is implicated in cancer fibrosis, we hypothesized that DDR1 interacts with NMIIA to enable collagen compaction by traction forces. Mechanical splinting of rat dermal wounds increased DDR1 expression and collagen alignment. In periodontal ligament of DDR1 knockout mice, collagen mechanical reorganization was reduced >30%. Similarly, cultured cells with DDR1 knockdown or expressing kinase-deficient DDR1d showed 50% reduction of aligned collagen. Tractional remodeling of collagen was dependent on DDR1 clustering, activation, and interaction of the DDR1 C-terminal kinase domain with NMIIA filaments. Collagen remodeling by traction forces, DDR1 tyrosine phosphorylation, and myosin light chain phosphorylation were increased on stiff versus soft substrates. Thus, DDR1 clustering, activation, and interaction with NMIIA filaments enhance the collagen tractional remodeling that is important for collagen compaction in fibrosis.

  14. Age-dependent susceptibility to phenobarbital-resistant neonatal seizures: role of chloride co-transporters

    Directory of Open Access Journals (Sweden)

    Seok Kyu eKang

    2015-05-01

    Full Text Available Ischemia in the immature brain is an important cause of neonatal seizures. Temporal evolution of acquired neonatal seizures and their response to anticonvulsants are of great interest, given the unreliability of the clinical correlates and poor efficacy of first-line anti-seizure drugs. The expression and function of the electroneutral chloride co-transporters KCC2 and NKCC1 influence the anti-seizure efficacy of GABAA-agonists. To investigate ischemia-induced seizure susceptibility and efficacy of the GABAA-agonist phenobarbital (PB, with NKCC1 antagonist bumetanide (BTN as an adjunct treatment, we utilized permanent unilateral carotid-ligation to produce acute ischemic-seizures in postnatal day 7, 10 and 12 CD1 mice. Immediate post-ligation video-electroencephalograms (EEGs quantitatively evaluated baseline and post-treatment seizure burdens. Brains were examined for stroke-injury and western blot analyses to evaluate the expression of KCC2 and NKCC1. Severity of acute ischemic seizures post-ligation was highest at P7. PB was an efficacious anti-seizure agent at P10 and P12, but not at P7. BTN failed as an adjunct, at all ages tested and significantly blunted PB-efficacy at P10. Significant acute post-ischemic downregulation of KCC2 was detected at all ages. At P7, males displayed higher age-dependent seizure susceptibility, associated with a significant developmental lag in their KCC2 expression. This study established a novel neonatal mouse model of PB-resistant seizures that demonstrates age/sex-dependent susceptibility. The age-dependent profile of KCC2 expression and its post-insult downregulation may underlie the PB-resistance reported in this model. Blocking NKCC1 with low-dose BTN following PB treatment failed to improve PB-efficacy.

  15. Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab

    OpenAIRE

    Fujii, Rika; Friedman, Eitan R.; Richards, Jacob; Tsang, Kwong Y.; Heery, Christopher R.; Schlom, Jeffrey; Hodge, James W.

    2016-01-01

    Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 ch...

  16. 25-Hydroxycholesterol promotes fibroblast-mediated tissue remodeling through NF-κB dependent pathway

    International Nuclear Information System (INIS)

    Ichikawa, Tomohiro; Sugiura, Hisatoshi; Koarai, Akira; Kikuchi, Takashi; Hiramatsu, Masataka; Kawabata, Hiroki; Akamatsu, Keiichiro; Hirano, Tsunahiko; Nakanishi, Masanori; Matsunaga, Kazuto; Minakata, Yoshiaki; Ichinose, Masakazu

    2013-01-01

    Abnormal structural alterations termed remodeling, including fibrosis and alveolar wall destruction, are important features of the pathophysiology of chronic airway diseases such as chronic obstructive pulmonary disease (COPD) and asthma. 25-hydroxycholesterol (25-HC) is enzymatically produced by cholesterol 25-hydorxylase (CH25H) in macrophages and is reported to be involved in the formation of arteriosclerosis. We previously demonstrated that the expression of CH25H and production of 25HC were increased in the lungs of COPD. However, the role of 25-HC in lung tissue remodeling is unknown. In this study, we investigated the effect of 25-HC on fibroblast-mediated tissue remodeling using human fetal lung fibroblasts (HFL-1) in vitro. 25-HC significantly augmented α-smooth muscle actin (SMA) (P 1 production (P 1 release. These results suggest that 25-HC could contribute to fibroblast-mediated lung tissue remodeling by promoting myofibroblast differentiation and the excessive release of extracellular matrix protein and MMPs via an NF-κB-TGF-β dependent pathway

  17. Coexistence of Cushing syndrome from functional adrenal adenoma and Addison disease from immune-mediated adrenalitis.

    Science.gov (United States)

    Colucci, Randall; Jimenez, Rafael E; Farrar, William; Malgor, Ramiro; Kohn, Leonard; Schwartz, Frank L

    2012-06-01

    A 56-year-old woman presented with an incidental adrenal adenoma and physical examination findings that included moderate obesity, a slight cervicothoracic fat pad ("buffalo hump"), increased supraclavicular fat pads, and white abdominal striae. Biochemical workup revealed elevated levels of 24-hour urinary free cortisol but normal serum morning cortisol and suppressed levels of corticotropin, suggestive of adrenal-dependent Cushing syndrome. The resected adrenal gland revealed macronodular cortical hyperplasia with a dominant nodule. Other findings included an absent cortisol response to corticotropin stimulation, presence of serum anti-21-hydroxylase antibodies, and mononuclear cell infiltration--consistent with adrenalitis. The findings represent, to the authors' knowledge, the first known case of a patient with coexistent functional cortisol-secreting macronodular adrenal tumor resulting in Cushing syndrome and immune-mediated adrenalitis resulting in Addison disease.

  18. Serum leptin is correlated to high turnover in osteoporosis.

    Science.gov (United States)

    Hipmair, Gunter; Böhler, Nikolaus; Maschek, Wilma; Soriguer, Federico; Rojo-Martínez, Gemma; Schimetta, Wolfgang; Pichler, Robert

    2010-01-01

    Clinical data have suggested that obesity protects against osteoporosis. Leptin, mainly secreted by white adipose tissue, might be involved by mediating an effect on bone metabolism. This study was conducted to investigate a possible relationship of leptin and bone turn-over in postmenopausal women with osteoporosis. We measured bone mineral density (BMD), serum leptin levels and markers of bone metabolism, including osteocalcin and cross-laps in 44 patients with osteoporosis. The main group consisted of 32 postmenopausal women. Mean serum leptin was 13.1 microg/L and showed no statistically significant difference to the levels measured in a collective of normal persons adjusted for age and BMI. When related to serum cross-laps as markers of bone resorption, a positive correlation (posteoporosis.

  19. GABAA receptor: Positive and negative allosteric modulators.

    Science.gov (United States)

    Olsen, Richard W

    2018-01-31

    gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane transport of GABA, the GABA receptors Type A (GABA A R) and Type B (GABA B R) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABA B R is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABA A R pharmacology, the topic of this article. GABA A R are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABA A R the targets of agonist depressants and antagonist convulsants, but most GABA A R drugs act at other (allosteric) binding sites on the GABA A R proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABA A R subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABA A R subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABA A R subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABA A R subtype-dependent extracellular domain sites. Thus GABA A R subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of

  20. Comparative radiochemical and radio-immunometrical study on trophoblast-dependent serum parameters under parturition, and radio-immunological detection of pregnancy-specific β1-glycoprotein [SP1] in early pregnancy

    International Nuclear Information System (INIS)

    Weber, K.A.E.

    1982-01-01

    77 deliveries were studied clinically and chemically bench-scale using data acquisition sheets applicable for computer operation. According to a catalog 30 normal, 32 abnormal and 15 births featuring EPH gestosis were clearly defined and classified. Radiochemical or radio-immunometrical methods were used to simultaneously analyse and evaluate statistically the following 'trophoblast-dependent serum parameters' in subpartem and postpartem maternal blood, and in aterialized or pooled venous-arterialized umbilicalchord blood in the case groups mentioned above: - 17 β-hydroxysteroid; NAD(P)-oxydoreductase (17 β-HSD; - free oestriol (fE 3 ); -β-subunit of Human Chorionic Gonadotropin (β-hCG); - Human Chorionic Somatomammatropin (hCS=hPL); - and pregnancy-specific β 1 -glycoprotein (SP 1 ). A systematic study was made for the statistical correlations between trophoblast - dependent serum parameters and plancental and neonatal findings as well as for possible correlations between the serum parameters in both the maternal and fetal blood distribution spaces. (orig./MG) [de

  1. Inflammatory mediators in human epilepsy : A systematic review and meta-analysis

    NARCIS (Netherlands)

    de Vries, Evelien E.; van den Munckhof, Bart; Braun, Kees P J; van Royen-Kerkhof, Annet; de Jager, Wilco; Jansen, Floor E.

    Background: Accumulating evidence suggests a role for inflammation in the pathophysiology of epilepsy. Methods: We performed a systematic review and meta-analysis of studies that investigated inflammatory mediators in human epilepsy. Studies reporting on inflammatory mediators in serum,

  2. Increased serum erythropoietin activity in rats following intrarenal injection of nickel subsulfide

    International Nuclear Information System (INIS)

    Hopfer, S.M.; Sunderman, F.W. Jr.; Fredrickson, T.N.; Morse, E.E.

    1979-01-01

    To investigate the pathopysiologic mechanisms of nickel-induced erythocytosis, serum erythropoietin activities were measured in (a) pooled serum from rats at 2 wk after intrarenal injection of αNi 3 S 2 (5 mg/rat), and (b) pooled serum from control rats at 2 wk after intrarenal injection of sterile NaCl vehicle (0.4 ml/rat). A sensitive erythropoietin bioassay was employed, which entailed repetitive administration of test serums to post-hypoxic polycythemic mice in divided doses (12 s.c. injections of 0.5 ml of serum at 6 h intervals for 3 da; total dose = 6 ml of serum/mouse). The erythropoietin detection limit was approx. = 20 I.U./liter of serum. In mice which received pooled serum from αNi 3 S 2 -treated rats, erythrocyte 59 Fe-uptake averaged 28% (S.D. +- 5) (vs 3.7 +- 1.1% in control rats; P 3 S 2 -treated rats averaged 130 I.U./liter (S.D. +- 18) (vs 27 +- 6 I.U./liter in control rats; P 3 S 2 is mediated by increased serum erythropoietin activity

  3. Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210.

    Science.gov (United States)

    Watanabe, M; Wallace, P K; Keler, T; Deo, Y M; Akewanlop, C; Hayes, D F

    1999-02-01

    MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcgammaRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcgammaRI is expressed on human monocytes, macrophages, and IFN-gamma activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions. Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit. Both BsAb MDX-210 (via FcgammaRI) and MoAb 520C9 (mouse IgG1, via FcgammaRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF. BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.

  4. Simultaneous radioimmunoassay for corticosterone and deoxycortisol in human serum: Sex differences in the mean serum concentrations

    International Nuclear Information System (INIS)

    Schoeneshoefer, M.; Harendt, H.; Vecsei, P.

    1977-01-01

    A specific radioimmunoassay is described which allows the simultaneous determination of serum corticosterone and deoxycortisol. The normal serum concentration of both steroids were found to be dependent on sex and menstrual cycle. Mean concentrations (+-S.D.) in males, females (follicular phase) and females (luteal phase) were 4210 +- 2170 ng/l, 2410 +- 1480 ng/l and 4390 +- 2320 ng/l for corticosterone and 499 +- 273 ng/l, 207 +- 152 ng/l and 335 +- 182 ng/l for deoxycortisol. After adrenal stimulation by corticotropin itself or by insulin induced hypoglycemia, the serum concentrations of corticosterone became significantly higher than those of deoxycortisol. After oral administration of dexamethasone serum concentrations of both steroids were suppressed to levels below the limit of the normal range. One hour after oral metyrapone administration at midnight, serum corticosterone decreased, while serum deoxycortisol increased by a factor of about five. After eight hours serum concentrations of both steroids were increased considerably. Corticosterone attained levels slightly higher than the normal range and deoxycortisol rose to levels which were higher than the normal mean concentrations by a factor of about 500. (orig./AS) [de

  5. Antibody-mediated immunity to the obligate intracellular bacterial pathogen Coxiella burnetii is Fc receptor- and complement-independent

    Directory of Open Access Journals (Sweden)

    Heinzen Robert A

    2009-05-01

    Full Text Available Abstract Background The obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonosis Q fever. The intracellular niche of C. burnetii has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect naïve animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR or complement contribute to Ab-mediated immunity (AMI to C. burnetii. Results Virulent C. burnetii infects and replicates within human dendritic cells (DC without inducing their maturation or activation. We investigated the effects of Ab opsonized C. burnetii on human monocyte-derived and murine bone marrow-derived DC. Infection of DC with Ab-opsonized C. burnetii resulted in increased expression of maturation markers and inflammatory cytokine production. Bacteria that had been incubated with naïve serum had minimal effect on DC, similar to virulent C. burnetii alone. The effect of Ab opsonized C. burnetii on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o compared to C57Bl/6 (B6 mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to C. burnetii in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to C. burnetii is also complement-independent. Conclusion Despite our data showing FcR-dependent stimulation of DC in vitro, Ab-mediated immunity to C. burnetii in vivo is FcR-independent. We also found that passive immunity to this pathogen is independent of

  6. Globular Adiponectin Inhibits the Apoptosis of Mesenchymal Stem Cells Induced by Hypoxia and Serum Deprivation via the AdipoR1-Mediated Pathway

    Directory of Open Access Journals (Sweden)

    Xia-Qiu Tian

    2016-02-01

    Full Text Available Background/Aims: Poor viability of transplanted mesenchymal stem cells (MSCs within the ischemic heart limits their therapeutic potential for cardiac repair. Globular adiponectin (gAPN exerts anti-apoptotic effects on several types of stem cells. Herein, we investigated the effect of gAPN on the MSCs against apoptosis induced by hypoxia and serum deprivation (H/SD. Methods: MSCs exposed to H/SD conditions were treated with different concentrations of gAPN. To identify the main type of receptor, MSCs were transfected with siRNA targeting adiponectin receptor 1 or 2 (AdipoR1 or AdipoR2. To elucidate the downstream pathway, MSCs were pre-incubated with AMPK inhibitor Compound C. Apoptosis, caspase-3 activity and mitochondrial membrane potential were evaluated. Results: H/SD-induced MSCs apoptosis and caspase-3 activation were attenuated by gAPN in a concentration-dependent manner. gAPN increased Bcl-2 and decreased Bax expressions. The loss of mitochondrial membrane potential induced by H/SD was also abolished by gAPN. The protective effect of gAPN was significantly attenuated after the knockdown of AdipoR1 rather than AdipoR2. Moreover, Compound C partly suppressed the anti-apoptotic effect of gAPN. Conclusions: gAPN inhibits H/SD-induced apoptosis in MSCs via AdipoR1-mediated pathway, possibly linked to the activation of AMPK. gAPN may be a novel survival factor for MSCs in the ischemic engraftment environment.

  7. Globular Adiponectin Inhibits the Apoptosis of Mesenchymal Stem Cells Induced by Hypoxia and Serum Deprivation via the AdipoR1-Mediated Pathway.

    Science.gov (United States)

    Tian, Xia-Qiu; Yang, Yue-Jin; Li, Qing; Huang, Pei-Sen; Li, Xiang-Dong; Jin, Chen; Qi, Kang; Jiang, Lei-Pei; Chen, Gui-Hao

    2016-01-01

    Poor viability of transplanted mesenchymal stem cells (MSCs) within the ischemic heart limits their therapeutic potential for cardiac repair. Globular adiponectin (gAPN) exerts anti-apoptotic effects on several types of stem cells. Herein, we investigated the effect of gAPN on the MSCs against apoptosis induced by hypoxia and serum deprivation (H/SD). MSCs exposed to H/SD conditions were treated with different concentrations of gAPN. To identify the main type of receptor, MSCs were transfected with siRNA targeting adiponectin receptor 1 or 2 (AdipoR1 or AdipoR2). To elucidate the downstream pathway, MSCs were pre-incubated with AMPK inhibitor Compound C. Apoptosis, caspase-3 activity and mitochondrial membrane potential were evaluated. H/SD-induced MSCs apoptosis and caspase-3 activation were attenuated by gAPN in a concentration-dependent manner. gAPN increased Bcl-2 and decreased Bax expressions. The loss of mitochondrial membrane potential induced by H/SD was also abolished by gAPN. The protective effect of gAPN was significantly attenuated after the knockdown of AdipoR1 rather than AdipoR2. Moreover, Compound C partly suppressed the anti-apoptotic effect of gAPN. gAPN inhibits H/SD-induced apoptosis in MSCs via AdipoR1-mediated pathway, possibly linked to the activation of AMPK. gAPN may be a novel survival factor for MSCs in the ischemic engraftment environment. © 2016 The Author(s) Published by S. Karger AG, Basel.

  8. Serum microRNA miR-206 is decreased in hyperthyroidism and mediates thyroid hormone regulation of lipid metabolism in HepG2 human hepatoblastoma cells.

    Science.gov (United States)

    Zheng, Yingjuan; Zhao, Chao; Zhang, Naijian; Kang, Wenqin; Lu, Rongrong; Wu, Huadong; Geng, Yingxue; Zhao, Yaping; Xu, Xiaoyan

    2018-04-01

    The actions of thyroid hormone (TH) on lipid metabolism in the liver are associated with a number of genes involved in lipogenesis and lipid metabolism; however, the underlying mechanisms through which TH impacts on lipid metabolism remain to be elucidated. The present study aimed to investigate the effects of hyperthyroidism on the serum levels of the microRNA (miR) miR‑206 and the role of miR‑206 on TH‑regulated lipid metabolism in liver cells. Serum was obtained from 12 patients diagnosed with hyperthyroidism and 10 healthy control subjects. Human hepatoblastoma (HepG2) cells were used to study the effects of triiodothyronine (T3) and miR‑206 on lipid metabolism. Expression of miR‑206 in serum and cells was determined by reverse transcription‑quantitative polymerase chain reaction analysis. Lipid accumulation in HepG2 cells was assessed with Oil Red O staining. Suppression or overexpression of miR‑206 was performed via transfection with a miR‑206 mimic or miR‑206 inhibitor. Serum miR‑206 was significantly decreased in patients with hyperthyroidism compared with euthyroid controls. Treatment of HepG2 cells with T3 led to reduced total cholesterol (TC) and triglyceride (TG) content, accompanied by reduced miR‑206 expression. Inhibition of endogenous miR‑206 expression decreased intracellular TG and TC content in HepG2 cells. By contrast, overexpression of miR‑206 in HepG2 partially prevented the reduction in TG content induced by treatment with T3. In conclusion, serum miR‑206 expression is reduced in patients with hyperthyroidism. In addition, miR‑206 is involved in T3‑mediated regulation of lipid metabolism in HepG2 cells, indicating a role for miR‑206 in thyroid hormone‑induced disorders of lipid metabolism in the liver.

  9. Novel time-dependent vascular actions of {delta}{sup 9}-tetrahydrocannabinol mediated by peroxisome proliferator-activated receptor gamma

    Energy Technology Data Exchange (ETDEWEB)

    O' Sullivan, Saoirse E [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Tarling, Elizabeth J [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Bennett, Andrew J [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Kendall, David A [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Randall, Michael D [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom)

    2005-11-25

    Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, {delta}{sup 9}-tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPAR{gamma}). In vitro, THC (10 {mu}M) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPAR{gamma} agonist rosiglitazone and was inhibited by the PPAR{gamma} antagonist GW9662 (1 {mu}M), but not the cannabinoid CB{sub 1} receptor antagonist AM251 (1 {mu}M). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPAR{gamma}, transiently expressed in combination with retinoid X receptor {alpha} and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 {mu}M). In vitro incubation with THC (1 or 10 {mu}M, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPAR{gamma} ligands. The present results provide strong evidence that THC is a PPAR{gamma} ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors.

  10. Association of testosterone and BDNF serum levels with craving during alcohol withdrawal.

    Science.gov (United States)

    Heberlein, Annemarie; Lenz, Bernd; Opfermann, Birgitt; Gröschl, Michael; Janke, Eva; Stange, Katrin; Groh, Adrian; Kornhuber, Johannes; Frieling, Helge; Bleich, Stefan; Hillemacher, Thomas

    2016-08-01

    Preclinical and clinical studies show associations between testosterone and brain-derived neurotrophic growth factor (BDNF) serum levels. BDNF and testosterone have been independently reported to influence alcohol consumption. Therefore, we aimed to investigate a possible interplay of testosterone and BDNF contributing to alcohol dependence. Regarding possible interplay of testosterone and BDNF and the activity of the hypothalamic pituitary axis (HPA), we included cortisol serum levels in our research. We investigated testosterone and BDNF serum levels in a sample of 99 male alcohol-dependent patients during alcohol withdrawal (day 1, 7, and 14) and compared them to a healthy male control group (n = 17). The testosterone serum levels were significantly (p BDNF serum levels (day 1: p = 0.008). In a subgroup of patients showing high cortisol serum levels (putatively mirroring high HPA activity), we found a significant association of BDNF and testosterone as well as with alcohol craving measured by the Obsessive and Compulsive Drinking Scale (OCDS). Our data suggest a possible association of BDNF and testosterone serum levels, which may be relevant for the symptomatology of alcohol dependence. Further studies are needed to clarify our results. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Minimizing the effects of multicollinearity in the polynomial regression of age relationships and sex differences in serum levels of pregnenolone sulfate in healthy subjects.

    Science.gov (United States)

    Meloun, Milan; Hill, Martin; Vceláková-Havlíková, Helena

    2009-01-01

    Pregnenolone sulfate (PregS) is known as a steroid conjugate positively modulating N-methyl-D-aspartate receptors on neuronal membranes. These receptors are responsible for permeability of calcium channels and activation of neuronal function. Neuroactivating effect of PregS is also exerted via non-competitive negative modulation of GABA(A) receptors regulating the chloride influx. Recently, a penetrability of blood-brain barrier for PregS was found in rat, but some experiments in agreement with this finding were reported even earlier. It is known that circulating levels of PregS in human are relatively high depending primarily on age and adrenal activity. Concerning the neuromodulating effect of PregS, we recently evaluated age relationships of PregS in both sexes using polynomial regression models known to bring about the problems of multicollinearity, i.e., strong correlations among independent variables. Several criteria for the selection of suitable bias are demonstrated. Biased estimators based on the generalized principal component regression (GPCR) method avoiding multicollinearity problems are described. Significant differences were found between men and women in the course of the age dependence of PregS. In women, a significant maximum was found around the 30th year followed by a rapid decline, while the maximum in men was achieved almost 10 years earlier and changes were minor up to the 60th year. The investigation of gender differences and age dependencies in PregS could be of interest given its well-known neurostimulating effect, relatively high serum concentration, and the probable partial permeability of the blood-brain barrier for the steroid conjugate. GPCR in combination with the MEP (mean quadric error of prediction) criterion is extremely useful and appealing for constructing biased models. It can also be used for achieving such estimates with regard to keeping the model course corresponding to the data trend, especially in polynomial type

  12. Comparison of the extraction and determination of serum exosome and miRNA in serum and the detection of miR-27a-3p in serum exosome of ALS patients.

    Science.gov (United States)

    Xu, Qian; Zhao, Yuying; Zhou, Xiaoyan; Luan, Jing; Cui, Yazhou; Han, Jinxiang

    2018-02-01

    Amyotrophic Lateral Sclerosis (ALS) is a muscle-bone degenerative disease, which lacks a specific index for diagnosis. In our previous studies, we found that exosomes mediated the interaction mechanism between muscle and bone at the cellular level, and myoblast exosomes can transfer miR-27a-3p to promote osteoblast mineralization. Therefore, we suppose that the expression of miR-27a-3p in the serum exosomes of ALS patients also changes. In this study, we used healthy human serum as a sample to find out the conditions and methods for extraction and detection. Then through comparison of the expression of miR-27a-3p in the serum exosomes of 10 ALS patients and healthy subjects, we found that in the ALS patients miR-27a-3p was down-regulated, and may be involved in the development of ALS, and therefore has potential as a reference for the diagnosis of ALS in the clinic.

  13. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller

    2008-01-01

    AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho......-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of c......AMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response....

  14. Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat

    DEFF Research Database (Denmark)

    Kiss, Alexander; Søderman, Andreas; Bundzikova, Jana

    2006-01-01

    Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immun...

  15. Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

    Directory of Open Access Journals (Sweden)

    Staffan Görander

    2014-11-01

    Full Text Available We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2 of herpes simplex virus 2 (HSV-2 in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1 and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC and complement-mediated cytolysis (ACMC activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

  16. Nootropic α7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators

    Science.gov (United States)

    Ng, Herman J.; Whittemore, Edward R.; Tran, Minhtam B.; Hogenkamp, Derk J.; Broide, Ron S.; Johnstone, Timothy B.; Zheng, Lijun; Stevens, Karen E.; Gee, Kelvin W.

    2007-01-01

    Activation of brain α7 nicotinic acetylcholine receptors (α7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of α7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective α7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-α-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at α7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of α7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction. PMID:17470817

  17. Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

    DEFF Research Database (Denmark)

    Elster, L; Kristiansen, U; Pickering, D S

    2001-01-01

    Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma......, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important...... for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed...

  18. Honokiol promotes non-rapid eye movement sleep via the benzodiazepine site of the GABA(A) receptor in mice.

    Science.gov (United States)

    Qu, Wei-Min; Yue, Xiao-Fang; Sun, Yu; Fan, Kun; Chen, Chang-Rui; Hou, Yi-Ping; Urade, Yoshihiro; Huang, Zhi-Li

    2012-10-01

    Decoctions of the Chinese herb houpu contain honokiol and are used to treat a variety of mental disorders, including depression. Depression commonly presents alongside sleep disorders and sleep disturbances, which appear to be a major risk factor for depression. Here, we have evaluated the somnogenic effect of honokiol and the mechanisms involved. Honokiol was administered i.p. at 20:00 h in mice. Flumazenil, an antagonist at the benzodiazepine site of the GABA(A) receptor, was administered i.p. 15 min before honokiol. The effects of honokiol were measured by EEG and electromyogram (EMG), c-Fos expression and in vitro electrophysiology. Honokiol (10 and 20 mg·kg⁻¹) significantly shortened the sleep latency to non-rapid eye movement (non-REM, NREM) sleep and increased the amount of NREM sleep. Honokiol increased the number of state transitions from wakefulness to NREM sleep and, subsequently, from NREM sleep to wakefulness. However, honokiol had no effect on either the amount of REM sleep or EEG power density of both NREM and REM sleep. Honokiol increased c-Fos expression in ventrolateral preoptic area (VLPO) neurons, as examined by immunostaining, and excited sleep-promoting neurons in the VLPO by whole-cell patch clamping in the brain slice. Pretreatment with flumazenil abolished the somnogenic effects and activation of the VLPO neurons by honokiol. Honokiol promoted NREM sleep by modulating the benzodiazepine site of the GABA(A) receptor, suggesting potential applications in the treatment of insomnia, especially for patients who experience difficulty in falling and staying asleep. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  19. Ultrasound-assisted interaction between chlorin-e6 and human serum albumin: pH dependence, singlet oxygen production, and formulation effect

    Science.gov (United States)

    Mocanu, Mihaela N.; Yan, Fei

    2018-02-01

    The interaction between chlorin e6 (Ce6) and human serum albumin (HSA) in the presence and absence of ultrasound have been investigated by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy. Ce6 is found to bind strongly to HSA at or near physiological pH conditions, but the strength of the binding is significantly weakened at lower pHs. The intrinsic fluorescence of HSA is incrementally quenched with increasing concentration of Ce6, and the quenching is enhanced after exposure to high-frequency ultrasound. Our experimental results suggest that Ce6-induced sonodynamic oxidation of HSA is mainly mediated by singlet oxygen. The formulation of Ce6 by high molecular weight polyvinylpyrrolidone (PVP) increased its stability in aqueous solutions and its quantum yield of singlet oxygen under ultrasound irradiation.

  20. Ursolic acid mediates photosensitization by initiating mitochondrial-dependent apoptosis

    Science.gov (United States)

    Lee, Yuan-Hao; Wang, Exing; Kumar, Neeru; Glickman, Randolph D.

    2013-02-01

    The signaling pathways PI3K/Akt and MAPK play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways may be modulated or inhibited by naturally-occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-kB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. Our current work indicates that UA causes these effects via the mTOR and insulin-mediated pathways. UA-modulated apoptosis, following exposure to UV radiation, is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. Flow cytometry analysis, DHE (dihydroethidium) staining and membrane permeability assay showed that UA pretreatment potentiated cell cycle arrest and radiation-induced apoptosis selectively on SM cells while DNA photo-oxidative damage (i.e. strand breakage) was reduced, presumably by some antioxidant activity of UA in RPE cells. The UA-mediated NF-κB activation in SM cells was reduced by rapamycin pretreatment, which indicates that these agents exert inter-antagonistic effects in the PI3K/Akt/mTOR pathway. In contrast, the antagonistic effect of UA on the PI3K/Akt pathway was reversed by insulin leading to greater NF-κB and p53 activation in RPE cells. MitoTracker, a mitochondrial functional assay, indicated that mitochondria in RPE cells experienced reduced oxidative stress while those in SM cells exhibited increased oxidative stress upon UA pretreatment. When rapamycin administration was followed by UA, mitochondrial oxidative stress was increased in RPE cells but decreased in SM cells. These results indicate that UA modulates p53 and NF-κB, initiating a mitogenic response to radiation that triggers mitochondria-dependent apoptosis.

  1. Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function

    International Nuclear Information System (INIS)

    Garg, Himanshu; Joshi, Anjali; Tompkins, Wayne A.

    2004-01-01

    Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function

  2. Studies on ADCC (antibody-dependent cell-mediated cytotoxicity) using sheep red blood cells as target cells, 2

    International Nuclear Information System (INIS)

    Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

    1979-01-01

    A non-specific cytotoxic mediator from effector cells (human peripheral blood leukocytes) was investigated in the ADCC (antibody-dependent cell-mediated cytotoxicity) system using antibody-coated sheep red blood cells (SRBC) as target cells. 51 Cr-labelled homologous (sheep) or heterologous (human) red blood cells were used as adjacent cells. Either crude lymphocyte fraction, phagocyte depleted fraction or granulocyte rich fraction separated from human peripheral leukocytes showed moderate cytotoxic effect on homologous adjacent cells, however no cytotoxic activity on heterologous adjacent cells was demonstrated in any leukocyte fraction. This suggests that the cytotoxic effects on homologous adjacent cells were resulted from the translocation of antibody molecules to adjacent cells from antibody-coated target cells. We concluded that the cytotoxic mechanism in this ADCC system was not mediated by non-specific soluble factors released from either human peripheral lymphocytes, monocytes or granulocytes. (author)

  3. Modeling Serum Creatinine in Septic ICU Patients

    DEFF Research Database (Denmark)

    De Gaetano, Andrea; Cortese, Giuliana; Pedersen, Morten Gram

    2004-01-01

    Serum creatinine is a metabolite assumed to be constantly produced by the normally functioning muscle mass and is a good measure for monitoring daily renal function in the intensive care unit (ICU). High serum creatinine levels or an abnormal departure from normal pre-disease basal levels....... The present work details the structure of a model describing observed creatinine serum concentration (CSC) variations, depending on the time-varying septic insult to renal function in ICU patients, as well as the estimation of its parameters. CSC determinations were routinely obtained from 12 patients...

  4. Tyr phosphatase-mediated P-ERK inhibition suppresses senescence in EIA + v-raf transformed cells, which, paradoxically, are apoptosis-protected in a MEK-dependent manner.

    Science.gov (United States)

    De Vitis, Stefania; Sonia Treglia, Antonella; Ulianich, Luca; Turco, Stefano; Terrazzano, Giuseppe; Lombardi, Angela; Miele, Claudia; Garbi, Corrado; Beguinot, Francesco; Di Jeso, Bruno

    2011-02-01

    Activation of the Ras-Raf-extracellular signal-regulated kinase (ERK) pathway causes not only proliferation and suppression of apoptosis but also the antioncogenic response of senescence. How these contrasting effects are reconciled to achieve cell transformation and cancer formation is poorly understood. In a system of two-step carcinogenesis (dedifferentiated PC EIA, transformed PC EIA-polyoma-middle T [PC EIA + Py] and PC EIA-v-raf [PC EIA + raf] cells], v-raf cooperated with EIA by virtue of a strong prosurvival effect, not elicited by Py-middle T, evident toward serum-deprivation-and H(2)O(2)-induced apoptosis. Apoptosis was detected by DNA fragmentation and annexin V staining. The prosurvival function of v-raf was, in part, mitogen-activated protein kinase/ERK kinase (MEK)-dependent, as shown by pharmacological MEK inhibition. The MEK-dependent antiapoptotic effect of v-raf was exerted despite a lower level of P-ERK1/2 in EIA + raf cells with respect to EIA + Py/EIA cells, which was dependent on a high tyrosine phosphatase activity, as shown by orthovanadate blockade. An ERK1/2 tyrosine phosphatase was likely involved. The high tyrosine phosphatase activity was instrumental to the complete suppression of senescence, detected by β-galactosidase activity, because tyrosine phosphatase blockade induced senescence in EIA + raf but not in EIA + Py cells. High tyrosine phosphatase activity and evasion from senescence were confirmed in an anaplastic thyroid cancer cell line. Therefore, besides EIA, EIA + raf cells suppress senescence through a new mechanism, namely, phosphatase-mediated P-ERK1/2 inhibition, but, paradoxically, retain the oncogenic effects of the Raf-ERK pathway. We propose that the survival effect of Raf is not a function of absolute P-ERK1/2 levels at a given time but is rather dynamically dependent on greater variations after an apoptotic stimulus.

  5. Tyr Phosphatase-Mediated P-ERK Inhibition Suppresses Senescence in EIA + v-raf Transformed Cells, Which, Paradoxically, Are Apoptosis-Protected in a MEK-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Stefania De Vitis

    2011-02-01

    Full Text Available Activation of the Ras-Raf-extracellular signal-regulated kinase (ERK pathway causes not only proliferation and suppression of apoptosis but also the antioncogenic response of senescence. How these contrasting effects are reconciled to achieve cell transformation and cancer formation is poorly understood. In a system of two-step carcinogenesis (dedifferentiated PC EIA, transformed PC EIA-polyoma-middle T [PC EIA + Py] and PC EIA-v-raf [PC EIA + raf] cells], v-raf cooperated with EIA by virtue of a strong prosurvival effect, not elicited by Py-middle T, evident toward serum-deprivation-and H2O2-induced apoptosis. Apoptosis was detected by DNA fragmentation and annexin V staining. The prosurvival function of v-raf was, in part, mitogen-activated protein kinase/ERK kinase (MEK-dependent, as shown by pharmacological MEK inhibition. The MEK-dependent antiapoptotic effect of v-raf was exerted despite a lower level of P-ERK1/2 in EIA + raf cells with respect to EIA + Py/EIA cells, which was dependent on a high tyrosine phosphatase activity, as shown by orthovanadate blockade. An ERK1/2 tyrosine phosphatase was likely involved. The high tyrosine phosphatase activity was instrumental to the complete suppression of senescence, detected by β-galactosidase activity, because tyrosine phosphatase blockade induced senescence in EIA + raf but not in EIA + Py cells. High tyrosine phosphatase activity and evasion from senescence were confirmed in an anaplastic thyroid cancer cell line. Therefore, besides EIA, EIA + raf cells suppress senescence through a new mechanism, namely, phosphatase-mediated P-ERK1/2 inhibition, but, paradoxically, retain the oncogenic effects of the Raf-ERK pathway. We propose that the survival effect of Raf is not a function of absolute P-ERK1/2 levels at a given time but is rather dynamically dependent on greater variations after an apoptotic stimulus.

  6. Zinc in human serum. Biochemical and clinical aspects. Zink i humant serum. Biokemiske og kliniske aspekter

    Energy Technology Data Exchange (ETDEWEB)

    Kiilerich, S

    1987-01-01

    The zinc ion is essential for the living organism. Many pathological conditions have been described as a consequence of zinc deficiency. As zinc constitutes less than 0.01 percent of the body weight, it conventionally belongs to the group of trace elements. The method of atomic absorption spectrophotometry is used to measure the concentration of zinc in serum and urine from healthy persons. The assumptions of the method is discussed. The importance of protein binding, diet and the diurnal variation of serum zinc concentration is presented. Serum versus plasma zinc concentration is discussed. Reference serum zinc values from 104 normal subjects are given. Zinc in serum is almost entirely bound to proteins. A preliminary model for the estimation of the distribution of zinc between serum albumin and ..cap alpha../sub 2/-macroglobulin is set up. This estimate has been examined by an ultracentrufugation method. The binding of zinc to a ..cap alpha../sup 2/-macroglobulin in normal persons is appoximately 7 percent, in patients with cirrhosis of the liver of alcoholic origin approximately 6 percent, in patients with insulin dependent diabetes mellitus approximately 5 percent, and in patients with chronic renal failure approximately 2 percent. It is concluded, therefore, that for clinical purposes it is sufficient to use the concentration of total serum zinc corrected for the concentration of serum albumin.

  7. 2.6. Sorption of serum albumin by ethynyl-piperidol hydrogels

    International Nuclear Information System (INIS)

    Khalikov, D.Kh.

    2012-01-01

    The sorption of serum albumin by ethynyl-piperidol hydrogels was studied in this article. Albumins adsorption on the surface of solids was considered. The capacity of cross-linked ethynyl piperidol polymers to the serum albumin was considered as well. The kinetic curves of sorption of human serum albumin by triple copolymer of isopropenyl trimethyl ethynyl piperidol were constructed. Sorption activity of ethynyl-piperidol polymers depending on ph of solution of human serum albumin were defined. Influence of solution ionic strength on sorption of human serum albumin was defined as well. The desorption of human serum albumin from the complexes with hydrogels was examined.

  8. Linking subordinate political skill to supervisor dependence and reward recommendations: a moderated mediation model.

    Science.gov (United States)

    Shi, Junqi; Johnson, Russell E; Liu, Yihao; Wang, Mo

    2013-03-01

    In this study, we examined the relations of subordinate political skill with supervisor's dependence on the subordinate and supervisor reward recommendation, as well as mediating (interaction frequency with supervisor) and moderating (supervisor political behavior) variables of these relations. Our theoretical model was tested using data collected from employees in a company that specialized in construction management. Analyses of multisource and lagged data from 53 construction management team supervisors and 296 subordinates indicated that subordinate political skill was positively related to supervisor reward recommendation via subordinate's interaction frequency with supervisor. Although interaction frequency with a supervisor was also positively related to the supervisor's dependence on the subordinate, the indirect effect of subordinate political skill on dependence was not significant. Further, both the relationship between subordinate political skill and interaction frequency with a supervisor and the indirect relationships between subordinate political skill and supervisor reward recommendation were stronger when supervisors exhibited more political behavior.

  9. Ratio-of-Mediator-Probability Weighting for Causal Mediation Analysis in the Presence of Treatment-by-Mediator Interaction

    Science.gov (United States)

    Hong, Guanglei; Deutsch, Jonah; Hill, Heather D.

    2015-01-01

    Conventional methods for mediation analysis generate biased results when the mediator--outcome relationship depends on the treatment condition. This article shows how the ratio-of-mediator-probability weighting (RMPW) method can be used to decompose total effects into natural direct and indirect effects in the presence of treatment-by-mediator…

  10. The anorexic agents, sibutramine and fenfluramine, depress GABAB-induced inhibitory postsynaptic potentials in rat mesencephalic dopaminergic cells

    Science.gov (United States)

    Ledonne, Ada; Sebastianelli, Luca; Federici, Mauro; Bernardi, Giorgio; Mercuri, Nicola Biagio

    2009-01-01

    Background and purpose Nutrition is the result of a complex interaction among environmental, homeostatic and reward-related processes. Accumulating evidence supports key roles for the dopaminergic neurons of the ventral midbrain in regulating feeding behaviour. For this reason, in the present study, we have investigated the electrophysiological effects of two centrally acting anorexic agents, fenfluramine and sibutramine, on these cells. Experimental approach Rat midbrain slices were used to make intracellular recordings from dopaminergic neurons of the substantia nigra and the ventral tegmental area. Gamma-aminobutyric acid (GABA)-mediated synaptic transmission was assessed from the inhibitory postsynaptic potentials (IPSPs) mediated by GABAA and GABAB receptors. Key results Fenfluramine and sibutramine reduced, concentration-dependently, the GABAB IPSPs, without affecting the GABAA-mediated potentials. This effect is presynaptic, as postsynaptic membrane responses induced by application of a GABAB receptor agonist, baclofen, were not affected by the two drugs. Furthermore, the selective 5-hydroxytriptamine 1B (5-HT1B) receptor antagonist, SB216641, blocked the reduction of GABAB IPSPs caused by fenfluramine and sibutramine, indicating that the receptor mediating this effect is 5-HT1B. Conclusions and implications Two anorexic agents, fenfluramine and sibutramine, induced the activation of 5-HT1B receptors located on presynaptic GABAergic terminals, thus reducing the release of GABA. This action can alter the strength of synaptic afferents that modify the activity of dopaminergic neurons, inducing neuronal excitation. Our results reveal an additional mechanism of action for fenfluramine and sibutramine that might contribute to reducing food intake, by influencing the pleasurable and motor aspects of feeding behaviour. PMID:19298257

  11. Theta oscillations at encoding mediate the context-dependent nature of human episodic memory.

    Science.gov (United States)

    Staudigl, Tobias; Hanslmayr, Simon

    2013-06-17

    Human episodic memory is highly context dependent. Therefore, retrieval benefits when a memory is recalled in the same context compared to a different context. This implies that items and contexts are bound together during encoding, such that the reinstatement of the initial context at test improves retrieval. Animal studies suggest that theta oscillations and theta-to-gamma cross-frequency coupling modulate such item-context binding, but direct evidence from humans is scarce. We investigated this issue by manipulating the overlap of contextual features between encoding and retrieval. Participants studied words superimposed on movie clips and were later tested by presenting the word with either the same or a different movie. The results show that memory performance and the oscillatory correlates of memory formation crucially depend on the overlap of the context between encoding and test. When the context matched, high theta power during encoding was related to successful recognition, whereas the opposite pattern emerged in the context-mismatch condition. In addition, cross-frequency coupling analysis revealed a context-dependent theta-to-gamma memory effect specifically in the left hippocampus. These results reveal for the first time that context-dependent episodic memory effects are mediated by theta oscillatory activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1-IKKϵ-IRF3 axis activation.

    Science.gov (United States)

    Tsukamoto, Hiroki; Takeuchi, Shino; Kubota, Kanae; Kobayashi, Yohei; Kozakai, Sao; Ukai, Ippo; Shichiku, Ayumi; Okubo, Misaki; Numasaki, Muneo; Kanemitsu, Yoshitomi; Matsumoto, Yotaro; Nochi, Tomonori; Watanabe, Kouichi; Aso, Hisashi; Tomioka, Yoshihisa

    2018-05-14

    Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-β expression. However, LPS-stimulated late activation of NFκB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-β pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

  13. The Prognostic Significance of The Serum Tumor Necrosis Factor (TNF-Related Apoptosis-Inducing Ligand (TRAIL in Childhood Acute Leukemias

    Directory of Open Access Journals (Sweden)

    Zeliha Haytoglu

    2015-12-01

    Results: The comparison of the average values of the TRAIL levels in acute leukemia patients and control group have shown that patients with leukemia have low serum TRAIL levels (p=0.002. In patients with high-risk-grade (HRG of ALL compared with control group have shown low serum TRAIL levels in HRG of ALL (p=0.008. In patients with common acute lymphoblastic leukemia antigen(CALLA(- B ALL compared with control group have shown low serum TRAIL levels in CALLA(- B ALL (p=0.004. Children with acute leukemias (ALL, AML who died during treatment compared with survived group have shown low levels of serum TRAIL in expired patients (p=0.004. Conclusion: As a result, serum TRAIL might play a role in leukomegenesis. The low levels of serum TRAIL detected in our patients may be associated with leukomogenezis and impaired TRAIL-mediated apoptosis. To suggest soluble TRAIL's role in acute leukemias detection of TRAIL-mediated apoptosis is needed. The low serum TRAIL may be used as a sign of bad prognosis. For more comphrensive results prospective studies with greaater number of patients are needed. [Cukurova Med J 2015; 40(4.000: 774-781

  14. Age- and sex-dependent susceptibility to phenobarbital-resistant neonatal seizures: role of chloride co-transporters.

    Science.gov (United States)

    Kang, Seok Kyu; Markowitz, Geoffrey J; Kim, Shin Tae; Johnston, Michael V; Kadam, Shilpa D

    2015-01-01

    Ischemia in the immature brain is an important cause of neonatal seizures. Temporal evolution of acquired neonatal seizures and their response to anticonvulsants are of great interest, given the unreliability of the clinical correlates and poor efficacy of first-line anti-seizure drugs. The expression and function of the electroneutral chloride co-transporters KCC2 and NKCC1 influence the anti-seizure efficacy of GABAA-agonists. To investigate ischemia-induced seizure susceptibility and efficacy of the GABAA-agonist phenobarbital (PB), with NKCC1 antagonist bumetanide (BTN) as an adjunct treatment, we utilized permanent unilateral carotid-ligation to produce acute ischemic-seizures in post-natal day 7, 10, and 12 CD1 mice. Immediate post-ligation video-electroencephalograms (EEGs) quantitatively evaluated baseline and post-treatment seizure burdens. Brains were examined for stroke-injury and western blot analyses to evaluate the expression of KCC2 and NKCC1. Severity of acute ischemic seizures post-ligation was highest at P7. PB was an efficacious anti-seizure agent at P10 and P12, but not at P7. BTN failed as an adjunct, at all ages tested and significantly blunted PB-efficacy at P10. Significant acute post-ischemic downregulation of KCC2 was detected at all ages. At P7, males displayed higher age-dependent seizure susceptibility, associated with a significant developmental lag in their KCC2 expression. This study established a novel neonatal mouse model of PB-resistant seizures that demonstrates age/sex-dependent susceptibility. The age-dependent profile of KCC2 expression and its post-insult downregulation may underlie the PB-resistance reported in this model. Blocking NKCC1 with low-dose BTN following PB treatment failed to improve PB-efficacy.

  15. Probing Temperature- and pH-Dependent Binding between Quantum Dots and Bovine Serum Albumin by Fluorescence Correlation Spectroscopy

    Directory of Open Access Journals (Sweden)

    Zonghua Wang

    2017-04-01

    Full Text Available Luminescent quantum dots (QDs with unique optical properties have potential applications in bio-imaging. The interaction between QDs and bio-molecules is important to the biological effect of QDs in vivo. In this paper, we have employed fluorescence correlation spectroscopy (FCS to probe the temperature- and pH-dependent interactions between CdSe QDs with carboxyl (QDs-COOH and bovine serum albumin (BSA in buffer solutions. The results have shown that microscopic dissociation constant K′D is in the range of (1.5 ± 0.2 × 10−5 to (8.6 ± 0.1 × 10−7 M, the Hill coefficient n is from 0.4 to 2.3, and the protein corona thickness is from 3.0 to 9.4 nm. Variable-temperature measurements have shown both negative values of ∆H and ∆S for BSA adsorption on QDs-COOH, while pH has a profound effect on the adsorption. Additional, FCS measurement QDs-COOH and proteins in whole mice serum and plasma samples has also been conducted. Finally, simulation results have shown four favored QD binding sites in BSA.

  16. Inflammatory impact of IFN-γ in CD8+ T cell-mediated lung injury is mediated by both Stat1-dependent and -independent pathways

    Science.gov (United States)

    Ramana, Chilakamarti V.; DeBerge, Matthew P.; Kumar, Aseem; Alia, Christopher S.; Durbin, Joan E.

    2015-01-01

    Influenza infection results in considerable pulmonary pathology, a significant component of which is mediated by CD8+ T cell effector functions. To isolate the specific contribution of CD8+ T cells to lung immunopathology, we utilized a nonviral murine model in which alveolar epithelial cells express an influenza antigen and injury is initiated by adoptive transfer of influenza-specific CD8+ T cells. We report that IFN-γ production by adoptively transferred influenza-specific CD8+ T cells is a significant contributor to acute lung injury following influenza antigen recognition, in isolation from its impact on viral clearance. CD8+ T cell production of IFN-γ enhanced lung epithelial cell expression of chemokines and the subsequent recruitment of inflammatory cells into the airways. Surprisingly, Stat1 deficiency in the adoptive-transfer recipients exacerbated the lung injury that was mediated by the transferred influenza-specific CD8+ T cells but was still dependent on IFN-γ production by these cells. Loss of Stat1 resulted in sustained activation of Stat3 signaling, dysregulated chemokine expression, and increased infiltration of the airways by inflammatory cells. Taken together, these data identify important roles for IFN-γ signaling and Stat1-independent IFN-γ signaling in regulating CD8+ T cell-mediated acute lung injury. This is the first study to demonstrate an anti-inflammatory effect of Stat1 on CD8+ T cell-mediated lung immunopathology without the complication of differences in viral load. PMID:25617378

  17. Hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries is mediated by intracellular methylglyoxal levels in a pathway dependent on oxidative stress

    DEFF Research Database (Denmark)

    Brouwers, O; Niessen, P M; Haenen, G

    2010-01-01

    -hydro-5-methylimidazolone (MG-H1) was detected with an antibody against MG-H1 and quantified with ultra-performance liquid chromatography (tandem) mass spectrometry. Reactive oxygen species formation was measured with a 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester probe...... for AGE ligand S100b did (p cells and adventitia by fivefold accompanied by an eightfold increase in the oxidative stress marker nitrotyrosine. Antioxidant pre-incubation prevented methylglyoxal......-induced impairment of vasoreactivity. CONCLUSIONS/INTERPRETATION: These data show that hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation is mediated by increased intracellular methylglyoxal levels in a pathway dependent on oxidative stress....

  18. Transmitters and pathways mediating inhibition of spinal itch-signaling neurons by scratching and other counterstimuli.

    Directory of Open Access Journals (Sweden)

    Tasuku Akiyama

    Full Text Available Scratching relieves itch, but the underlying neural mechanisms are poorly understood. We presently investigated a role for the inhibitory neurotransmitters GABA and glycine in scratch-evoked inhibition of spinal itch-signaling neurons in a mouse model of chronic dry skin itch. Superficial dorsal horn neurons ipsilateral to hindpaw dry skin treatment exhibited a high level of spontaneous firing that was significantly attenuated by cutaneous scratching, pinch and noxious heat. Scratch-evoked inhibition was nearly abolished by spinal delivery of the glycine antagonist, strychnine, and was markedly attenuated by respective GABA(A and GABA(B antagonists bicuculline and saclofen. Scratch-evoked inhibition was also significantly attenuated (but not abolished by interruption of the upper cervical spinal cord, indicating the involvement of both segmental and suprasegmental circuits that engage glycine- and GABA-mediated inhibition of spinal itch-signaling neurons by noxious counterstimuli.

  19. Time-Dependent Vascular Effects of Endocannabinoids Mediated by Peroxisome Proliferator-Activated Receptor Gamma (PPAR

    Directory of Open Access Journals (Sweden)

    Saoirse E. O'Sullivan

    2009-01-01

    Full Text Available The aim of the present study was to examine whether endocannabinoids cause PPAR-mediated vascular actions. Functional vascular studies were carried out in rat aortae. Anandamide and N-arachidonoyl-dopamine (NADA, but not palmitoylethanolamide, caused significant vasorelaxation over time (2 hours. Vasorelaxation to NADA, but not anandamide, was inhibited by CB1 receptor antagonism (AM251, 1 M, and vasorelaxation to both anandamide and NADA was inhibited by PPAR antagonism (GW9662, 1 M. Pharmacological inhibition of de novo protein synthesis, nitric oxide synthase, and super oxide dismutase abolished the responses to anandamide and NADA. Removal of the endothelium partly inhibited the vasorelaxant responses to anandamide and NADA. Inhibition of fatty acid amide hydrolase (URB597, 1 M inhibited the vasorelaxant response to NADA, but not anandamide. These data indicate that endocannabinoids cause time-dependent, PPAR-mediated vasorelaxation. Activation of PPAR in the vasculature may represent a novel mechanism by which endocannabinoids are involved in vascular regulation.

  20. Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    International Nuclear Information System (INIS)

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-01-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [ 3 H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [ 3 H]flunitrazepam and [ 3 H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [ 3 H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy

  1. Insulin-Independent GABAA Receptor-Mediated Response in the Barrel Cortex of Mice with Impaired Met Activity.

    Science.gov (United States)

    Lo, Fu-Sun; Erzurumlu, Reha S; Powell, Elizabeth M

    2016-03-30

    Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic variants, susceptibility alleles, and environmental perturbations. The autism associated geneMETtyrosine kinase has been implicated in many behavioral domains and endophenotypes of autism, including abnormal neural signaling in human sensory cortex. We investigated somatosensory thalamocortical synaptic communication in mice deficient in Met activity in cortical excitatory neurons to gain insights into aberrant somatosensation characteristic of ASD. The ratio of excitation to inhibition is dramatically increased due to decreased postsynaptic GABAAreceptor-mediated inhibition in the trigeminal thalamocortical pathway of mice lacking active Met in the cerebral cortex. Furthermore, in contrast to wild-type mice, insulin failed to increase GABAAreceptor-mediated response in the barrel cortex of mice with compromised Met signaling. Thus, lacking insulin effects may be a risk factor in ASD pathogenesis. A proposed common cause of neurodevelopmental disorders is an imbalance in excitatory neural transmission, provided by the glutamatergic neurons, and the inhibitory signals from the GABAergic interneurons. Many genes associated with autism spectrum disorders impair synaptic transmission in the expected cell type. Previously, inactivation of the autism-associated Met tyrosine kinase receptor in GABAergic interneurons led to decreased inhibition. In thus report, decreased Met signaling in glutamatergic neurons had no effect on excitation, but decimated inhibition. Further experiments indicate that loss of Met activity downregulates GABAAreceptors on glutamatergic neurons in an insulin independent manner. These data provide a new mechanism for the loss of inhibition and subsequent abnormal excitation/inhibition balance and potential molecular candidates for treatment or prevention. Copyright © 2016 the authors 0270-6474/16/363691-07$15.00/0.

  2. Conglutinin exhibits a complement-dependent enhancement of the respiratory burst of phagocytes stimulated by E. coli

    DEFF Research Database (Denmark)

    Friis, P; Svehag, S E; Andersen, Ove

    1991-01-01

    . Conglutinin enhances, in a dose-dependent manner, the respiratory burst of spleen cells stimulated with serum-opsonized Escherichia coli. The enhancement was only demonstrable in the presence of a functional complement system. The conglutinin-mediated enhancement of the respiratory burst was inhibited......Conglutinin is a mammalian C-type lectin which shows anti-bacterial activity when tested in vivo and in vitro. This study concerns the effect of conglutinin on the respiratory burst of murine spleen cells, using a chemiluminescence assay for measurement of generated reactive oxygen metabolites...

  3. Temperature dependent rapid annealing effect induces amorphous aggregation of human serum albumin.

    Science.gov (United States)

    Ishtikhar, Mohd; Ali, Mohd Sajid; Atta, Ayman M; Al-Lohedan, Hammad; Badr, Gamal; Khan, Rizwan Hasan

    2016-01-01

    This study represents an analysis of the thermal aggregation of human serum albumin (HSA) induced by novel rosin modified compounds. The aggregation process causes conformational alterations in the secondary and tertiary structures of the proteins. The conversion of globular protein to amorphous aggregates was carried out by spectroscopic, calorimetric and microscopic techniques to investigate the factors that are responsible for the structural, conformational and morphological alteration in the protein. Our outcome results show that the aggregation of HSA was dependent on the hydrophobicity, charge and temperature, because the formation of amorphous aggregates occurs in the presence of a novel cationic rosin compound, quaternary amine of rosin diethylaminoethyl ester (QRMAE), at 40°C and pH 7.4 (but at 25°C on similar pH value, there was no evidence of aggregate formation). In addition, the parent compound of QRMAE, i.e., abietic acid, and other derivatives such as nonionic rosin compounds [(RMPEG-750) and (RMA-MPEG-750)] do not shows the aggregating property. This work provides precise and necessary information that aid in the understanding the effects of rosin derivative compounds on HSA. This study also restrains important information for athletes, health providers, pharmaceutical companies, industries, and soft drink-processing companies. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Loss of Ethanol Conditioned Taste Aversion and Motor Stimulation in Knockin Mice with Ethanol-Insensitive α2-Containing GABAA Receptors

    Science.gov (United States)

    Borghese, C. M.; McCracken, M. L.; Benavidez, J. M.; Geil, C. R.; Osterndorff-Kahanek, E.; Werner, D. F.; Iyer, S.; Swihart, A.; Harrison, N. L.; Homanics, G. E.; Harris, R. A.

    2011-01-01

    GABA type A receptors (GABAA-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABAA-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705–714, 2004; Pharmacol Biochem Behav 90:95–104, 2008; J Psychiatr Res 42:184–191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism. PMID:20876231

  5. On the length dependence of bridge-mediated electron transfer reactions

    International Nuclear Information System (INIS)

    Petrov, E.G.; Shevchenko, Ye.V.; May, V.

    2003-01-01

    Bridge-mediated nonadiabatic donor-acceptor (D-A) electron transfer (ET) is studied for the case of a regular molecular bridge of N identical units. It is shown that the multi-exponential ET kinetics reduces to a single-exponential transfer if, and only if, the integral population of the bridge remains small (less than 10 -2 ). An analytical expression for the overall D-A ET rate is derived and the necessary and sufficient conditions are formulated at which the rate is given as a sum of a superexchange and a sequential contribution. To describe experimental data on the N-dependence of ET reactions an approximate form of the overall transfer rate is derived. This expression is used to reproduce experimental data on distant ET through polyproline chains. Finally it is noted that the obtained analytical results can also be used for the description of more complex two-electron transfer reactions if the latter comprises separate single-electron pathways

  6. Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease.

    Science.gov (United States)

    Ahmad, Faraz; Singh, Kunal; Das, Debajyoti; Gowaikar, Ruturaj; Shaw, Eisha; Ramachandran, Arathy; Rupanagudi, Khader Valli; Kommaddi, Reddy Peera; Bennett, David A; Ravindranath, Vijayalakshmi

    2017-12-01

    Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD. Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APP Swe /PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9-10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains. Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity. We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269-1280.

  7. Increased Eotaxin and MCP-1 Levels in Serum from Individuals with Periodontitis and in Human Gingival Fibroblasts Exposed to Pro-Inflammatory Cytokines

    Science.gov (United States)

    Sulniute, Rima; Palmqvist, Py; Majster, Mirjam; Holm, Cecilia Koskinen; Zwicker, Stephanie; Clark, Reuben; Önell, Sebastian; Johansson, Ingegerd; Lerner, Ulf H.; Lundberg, Pernilla

    2015-01-01

    Periodontitis is a chronic inflammatory disease of tooth supporting tissues resulting in periodontal tissue destruction, which may ultimately lead to tooth loss. The disease is characterized by continuous leukocyte infiltration, likely mediated by local chemokine production but the pathogenic mechanisms are not fully elucidated. There are no reliable serologic biomarkers for the diagnosis of periodontitis, which is today based solely on the degree of local tissue destruction, and there is no available biological treatment tool. Prompted by the increasing interest in periodontitis and systemic inflammatory mediators we mapped serum cytokine and chemokine levels from periodontitis subjects and healthy controls. We used multivariate partial least squares (PLS) modeling and identified monocyte chemoattractant protein-1 (MCP-1) and eotaxin as clearly associated with periodontitis along with C-reactive protein (CRP), years of smoking and age, whereas the number of remaining teeth was associated with being healthy. Moreover, body mass index correlated significantly with serum MCP-1 and CRP, but not with eotaxin. We detected higher MCP-1 protein levels in inflamed gingival connective tissue compared to healthy but the eotaxin levels were undetectable. Primary human gingival fibroblasts displayed strongly increased expression of MCP-1 and eotaxin mRNA and protein when challenged with tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β), key mediators of periodontal inflammation. We also demonstrated that the upregulated chemokine expression was dependent on the NF-κΒ pathway. In summary, we identify higher levels of CRP, eotaxin and MCP-1 in serum of periodontitis patients. This, together with our finding that both CRP and MCP-1 correlates with BMI points towards an increased systemic inflammatory load in patients with periodontitis and high BMI. Targeting eotaxin and MCP-1 in periodontitis may result in reduced leukocyte infiltration and inflammation in

  8. Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

    Science.gov (United States)

    Stępiński, Dariusz

    2016-08-01

    Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development.

  9. Normal human serum (HS) prevents oxidant-induced lysis of cultured endothelial cells (ECs)

    International Nuclear Information System (INIS)

    Callahan, K.S.; Harlan, J.M.

    1986-01-01

    Most studies demonstrating oxidant lysis of cultured ECs are performed in serum-free media or media containing low concentrations of bovine serum. The authors found that HS protects human and bovine ECs from lysis caused by reagent H 2 O 2 or glucose/glucose oxidase (GO)-generated H 2 O 2 . EC injury was assessed by 51 Cr release, cell detachment, or trypan blue dye exclusion. Protective HS activity was dose-dependent with concentrations greater than or equal to 25% preventing lethal injury. Cytotoxicity at 24 hrs, induced by 20 mU/ml GO, was 90.1 +/- 5.2% without HS vs 1.7 +/- 4.6% with 25% HS present (20 exp). Similar protection was observed with heparinized plasma. Of note, comparable concentrations of bovine serum were devoid of protective activity. Addition of fatty acid-free albumin to the media was also without protective effect. Preliminary characterization showed HS activity was stable to 60 0 C for 30 min, non-dialyzable at 25,000 MW cutoff, and retained in delipidated serum. The HS protection was not merely due to scavenging of exogenous H 2 O 2 as A23187-induced EC lysis was also prevented by HS. Protective activity was not reproduced by purified cerruloplasmin or transferrin. In conclusion, unidentified factor(s) present in HS protect cultured ECs from oxidant-induced lysis. Since endothelium is normally exposed to 100% plasma, the authors suggest that in vitro studies of oxidant-mediated injury be performed in the presence of HS. Factor(s) in HS may play an important role in modulating oxidant-induced vascular injury in vivo

  10. Demonstration of the dynamic mass redistribution label-free technology as a useful cell-based pharmacological assay for endogenously expressed GABAA receptors

    DEFF Research Database (Denmark)

    Klein, Anders Bue; Nittegaard-Nielsen, Mia; Christensen, Julie T.

    2015-01-01

    the immortalized IMR-32 neuroblastoma cell line, which expresses relatively high levels of several endogenous GABAA receptor subunits, we show that GABA produces concentration-dependent cellular responses that can be measured and quantified in real-time. With the aid of the GABAA receptor-specific agonist muscimol...

  11. Serum apolipoprotein E concentration and polymorphism influence serum lipid levels in Chinese Shandong Han population.

    Science.gov (United States)

    Han, ShuYi; Xu, YiHui; Gao, MeiHua; Wang, YunShan; Wang, Jun; Liu, YanYan; Wang, Min; Zhang, XiaoQian

    2016-12-01

    Apolipoprotein E (ApoE), which has been shown to influence serum lipid parameters, can bind to multiple types of lipids and plays an important role in the metabolism and homeostasis of lipids and lipoproteins. A previous study showed that ApoE concentration significantly affects serum lipid levels independently of ApoE polymorphism. The serum lipid levels were also closely correlated with dietary habits, and Shandong cuisine is famous for its high salt and oil contents, which widely differ among the different areas in China. Therefore, studying the effect of ApoE polymorphism on ApoE concentration and serum lipid levels in Shandong province is very important.A total of 815 subjects including 285 men and 530 women were randomly selected and studied from Jinan, Shandong province. In order to evaluate the association of ApoE polymorphism and serum level on lipid profiles, the ApoE genotypes, as well as levels of fasting serum ApoE and other lipid parameters, were detected in all subjects.The frequency of the ApoE E3 allele was highest (83.1%), while those of E2 and E4 were 9.4% and 7.5%, respectively, which are similar to those in other Asian populations. ApoE2 allele carriers showed significantly increased ApoE levels but lower levels of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and Apolipoprotein B (ApoB).We found that ApoE level is influenced by ApoE polymorphism in a gene-dependent manner. The ApoE polymorphism showed different influences on serum lipid parameters with increasing age and body mass index (BMI) in our Shandong Han population.

  12. In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

    Science.gov (United States)

    Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

    2005-06-01

    Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the

  13. The Mediational Role of Relational Psychological Contract in Belief in a Zero-Sum Game and Work Input Attitude Dependency

    Directory of Open Access Journals (Sweden)

    Adamska Krystyna

    2015-12-01

    Full Text Available The purpose of this study was to investigate the mediational role of relational psychological contract in social beliefs and work input attitude dependency. We analyzed data taken from employees (N = 258 in four different organizations operating in the Pomeranian market.

  14. Listeria monocytogenes alters mast cell phenotype, mediator and osteopontin secretion in a listeriolysin-dependent manner.

    Directory of Open Access Journals (Sweden)

    Catherine E Jobbings

    Full Text Available Whilst mast cells participate in the immune defence against the intracellular bacterium Listeria monocytogenes, there is conflicting evidence regarding the ability of L. monocytogenes to infect mast cells. It is known that the pore-forming toxin listeriolysin (LLO is important for mast cell activation, degranulation and the release of pro-inflammatory cytokines. Mast cells, however, are a potential source of a wide range of cytokines, chemokines and other mediators including osteopontin, which contributes to the clearing of L. monocytogenes infections in vivo, although its source is unknown. We therefore aimed to resolve the controversy of mast cell infection by L. monocytogenes and investigated the extent of mediator release in response to the bacterium. In this paper we show that the infection of bone marrow-derived mast cells by L. monocytogenes is inefficient and LLO-independent. LLO, however, is required for calcium-independent mast cell degranulation as well as for the transient and selective downregulation of cell surface CD117 (c-kit on mast cells. We demonstrate that in addition to the key pro-inflammatory cytokines TNF-α and IL-6, mast cells release a wide range of other mediators in response to L. monocytogenes. Osteopontin, IL-2, IL-4, IL-13 and granulocyte macrophage colony-stimulating factor (GM-CSF, and chemokines including CCL2, CCL3, CCL4 and CCL5 are released in a MyD88-dependent manner. The wide range of mediators released by mast cells in response to L. monocytogenes may play an important role in the recruitment and activation of a variety of immune cells in vivo. The cocktail of mediators, however, is unlikely to skew the immune response to a particular effector response. We propose that mast cells provide a hitherto unreported source of osteopontin, and may provide an important role in co-ordinating the immune response during Listeria infection.

  15. Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

    Science.gov (United States)

    Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

    2014-03-01

    (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

  16. Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP.

    Directory of Open Access Journals (Sweden)

    Amanda E Calvert

    Full Text Available Zika virus (ZIKV has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/μl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV and chikungunya virus (CHIKV. The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise.

  17. GABAergic actions on cholinergic laterodorsal tegmental neurons

    DEFF Research Database (Denmark)

    Kohlmeier, K A; Kristiansen, Uffe

    2010-01-01

    Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated....... In vivo studies suggest that GABAergic mechanisms within the pons play a critical role in behavioral state switching. However, the postsynaptic, electrophysiological actions of GABA on LDT neurons, as well as the identity of GABA receptors present in the LDT mediating these actions is virtually unexplored...... neurons. Post-synaptic location of GABA(A) receptors was demonstrated by persistence of muscimol-induced inward currents in TTX and low Ca(2+) solutions. THIP, a selective GABA(A) receptor agonist with a preference for d-subunit containing GABA(A) receptors, induced inward currents, suggesting...

  18. Cryptochrome mediates light-dependent magnetosensitivity of Drosophila's circadian clock.

    Directory of Open Access Journals (Sweden)

    Taishi Yoshii

    2009-04-01

    Full Text Available Since 1960, magnetic fields have been discussed as Zeitgebers for circadian clocks, but the mechanism by which clocks perceive and process magnetic information has remained unknown. Recently, the radical-pair model involving light-activated photoreceptors as magnetic field sensors has gained considerable support, and the blue-light photoreceptor cryptochrome (CRY has been proposed as a suitable molecule to mediate such magnetosensitivity. Since CRY is expressed in the circadian clock neurons and acts as a critical photoreceptor of Drosophila's clock, we aimed to test the role of CRY in magnetosensitivity of the circadian clock. In response to light, CRY causes slowing of the clock, ultimately leading to arrhythmic behavior. We expected that in the presence of applied magnetic fields, the impact of CRY on clock rhythmicity should be altered. Furthermore, according to the radical-pair hypothesis this response should be dependent on wavelength and on the field strength applied. We tested the effect of applied static magnetic fields on the circadian clock and found that flies exposed to these fields indeed showed enhanced slowing of clock rhythms. This effect was maximal at 300 muT, and reduced at both higher and lower field strengths. Clock response to magnetic fields was present in blue light, but absent under red-light illumination, which does not activate CRY. Furthermore, cry(b and cry(OUT mutants did not show any response, and flies overexpressing CRY in the clock neurons exhibited an enhanced response to the field. We conclude that Drosophila's circadian clock is sensitive to magnetic fields and that this sensitivity depends on light activation of CRY and on the applied field strength, consistent with the radical pair mechanism. CRY is widespread throughout biological systems and has been suggested as receptor for magnetic compass orientation in migratory birds. The present data establish the circadian clock of Drosophila as a model system

  19. Imaging TCR-Dependent NFAT-Mediated T-Cell Activation with Positron Emission Tomography In Vivo

    Directory of Open Access Journals (Sweden)

    Vladimir Ponomarev

    2001-01-01

    Full Text Available A noninvasive method for molecular imaging of T-cell activity in vivo would be of considerable value. It would aid in understanding the role of specific genes and signal transduction pathways in the course of normal and pathologic immune responses, could elucidate temporal dynamics and immune regulation at different stages of disease and following therapy. We developed and assessed a novel method for monitoring the T-cell receptor (TCR -dependent nuclear factor of activated T cells (NFAT -mediated activation of T cells by optical fluorescence imaging (OFI and positron emission tomography (PET. The herpes simplex virus type 1 thymidine kinase/green fluorescent protein [HSV1-tk/GFP (TKGFP ] dual reporter gene was used to monitor NFAT-mediated transcriptional activation in human Jurkat cells. A recombinant retrovirus bearing the NFAT-TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting NFAT-specific enhancer. Transduced Jurkat cells were used to establish subcutaneous infiltrates in nude rats. We demonstrated that noninvasive OR and nuclear imaging of T-cell activation is feasible using the NFAT-TKGFP reporter system. PET imaging with [124]FIAU using the NFAT-TKGFP reporter system is sufficiently sensitive to detect T-cell activation in vivo. PET images were confirmed by independent measurements of T-cell activation (e.g., CD69 and induction of GFP fluorescence. PET imaging of TCR-induced NFAT-dependent transcriptional activity may be useful in the assessment of T cell responses, T-cell-based adoptive therapies, vaccination strategies and immunosuppressive drugs.

  20. Significance of serum and urine β2-MG abnormality for diabetes

    International Nuclear Information System (INIS)

    Yu Fengling; Zhang Qiliang; Feng Zhixu; Hou Xiangzhen

    1995-01-01

    Levels of serum and urine β 2 -MG are determined in 114 patients with diabetes. It is found that levels of serum and urine β 2 -MG in diabetes are significantly higher than that of normal contrasts (P 2 -MG are increased with diabetes progress, especially urine β 2 -MG. There is no difference in levels of serum and urine β 2 -MG between non-insulin dependent diabetes mellitus (NIDDM) and insulin dependent diabetes mellitus (IDDM, P>0.05). Urine β 2 -MG levels of diabetes are relatively increased with the increase of serum β 2 -MG levels. Both are obviously positive correlation. While diabetes progressing, both are gradually increased. It can be shown that the longer diabetes process, the more renal function was damaged. Therefore, determination of β 2 -MG is very important for early diagnosing, preventing and treating diabetic nephropathy

  1. Stimulus-dependent modulation of spike burst length in cat striate cortical cells.

    Science.gov (United States)

    DeBusk, B C; DeBruyn, E J; Snider, R K; Kabara, J F; Bonds, A B

    1997-07-01

    Burst activity, defined by groups of two or more spikes with intervals of cats. Bursting varied broadly across a population of 507 simple and complex cells. Half of this population had > or = 42% of their spikes contained in bursts. The fraction of spikes in bursts did not vary as a function of average firing rate and was stationary over time. Peaks in the interspike interval histograms were found at both 3-5 ms and 10-30 ms. In many cells the locations of these peaks were independent of firing rate, indicating a quantized control of firing behavior at two different time scales. The activity at the shorter time scale most likely results from intrinsic properties of the cell membrane, and that at the longer scale from recurrent network excitation. Burst frequency (bursts per s) and burst length (spikes per burst) both depended on firing rate. Burst frequency was essentially linear with firing rate, whereas burst length was a nonlinear function of firing rate and was also governed by stimulus orientation. At a given firing rate, burst length was greater for optimal orientations than for nonoptimal orientations. No organized orientation dependence was seen in bursts from lateral geniculate nucleus cells. Activation of cortical contrast gain control at low response amplitudes resulted in no burst length modulation, but burst shortening at optimal orientations was found in responses characterized by supersaturation. At a given firing rate, cortical burst length was shortened by microinjection of gamma-aminobutyric acid (GABA), and bursts became longer in the presence of N-methyl-bicuculline, a GABA(A) receptor blocker. These results are consistent with a model in which responses are reduced at nonoptimal orientations, at least in part, by burst shortening that is mediated by GABA. A similar mechanism contributes to response supersaturation at high contrasts via recruitment of inhibitory responses that are tuned to adjacent orientations. Burst length modulation can serve

  2. Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

    Directory of Open Access Journals (Sweden)

    Cunyou Zhao

    Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

  3. Serum concentration of IL-6, IL-2, TNF-α, and IFNγ in Vitiligo patients

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    Suman Singh

    2012-01-01

    Full Text Available Background: Vitiligo is an acquired depigmenting disorder characterized by the loss of functional melanocytes from the epidermis. Although the etiology of vitiligo is unknown, over the last few years, substantial data from clinical research has greatly supported the ′Autoimmune theory′ and this is supported by the frequent association of vitiligo with disorders that have an autoimmune origin, including Hashimoto′s thyroiditis, Graves disease, type 1 insulin-dependent diabetes mellitus, and Addison′s disease. As cytokines are important mediators of immunity, there is evidence to suggest that they play a major role in the pathogenesis of autoimmune diseases. Aim: Keeping this in view we have assayed sera for cytokine IL-6, IL-2, Tumor necrosis factor (TNF-α, and IFNγ in 80 cases of vitiligo and compared it with healthy subjects, in order to find out whether they play a role in the pathogenesis of vitiligo or not. Materials and Methods: Serum IL-6, IL-2, TNF-α, and IFNγ were done by the indirect enzyme linked immunosorbent assay (ELISA. Results: The mean serum IL-6 and IL-2 levels in the patient group were significantly higher when compared with those of the normal controls. The mean serum IFNγ level in patients with vitiligo was significantly lower than that in the control group. There was no significant difference in the serum level of TNF-α between vitiligo and healthy controls. Conclusion : An increase in the production of proinflammatory cytokines such as IL-6 and IL-2 in vitiligo patients may play an important role in melanocytic cytotoxicity. Thus, we speculate that the cytokine production of epidermal microenvironment may be involved in vitiligo.

  4. Serum Cytokine Profiles in Children with Crohn’s Disease

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    Ekaterina Vasilyeva

    2016-01-01

    Full Text Available Crohn’s disease (CD is a chronic inflammatory bowel disease that can be diagnosed at any age. There are two major patient groups based on diagnosis of this disease, before or after the age of 20 (juvenile/adolescent or adult, with disease progression in adults usually milder than in juvenile CD patients. Immune mechanisms have been suggested to play an important role in CD pathogenesis, with cytokines governing the development of the immune response. Upregulation of inflammatory cytokines in serum of juvenile and adult CD patients has been documented; still little is known about age-dependent differences in serum cytokine profiles of CD patients. We applied multiplex technology to analyze serum levels of 12 cytokines in juveniles and adults. We show that during the acute stage of the disease all CD patients have high serum levels of CXCL10, which remains upregulated during remission. Increased serum levels of TNF-α and IL-6 during the acute stage was characteristic of juvenile CD patients, whereas adult CD patients had upregulated levels of GM-CSF and IFN-γ. Taken together, these results demonstrate age-dependent differences in cytokine profiles, which may affect the pathogenesis of CD in patients at different ages of disease onset.

  5. GPER1-mediated IGFBP-1 induction modulates IGF-1-dependent signaling in tamoxifen-treated breast cancer cells.

    Science.gov (United States)

    Vaziri-Gohar, Ali; Houston, Kevin D

    2016-02-15

    Tamoxifen, a selective estrogen receptor modulator, is a commonly prescribed adjuvant therapy for estrogen receptor-α (ERα)-positive breast cancer patients. To determine if extracellular factors contribute to the modulation of IGF-1 signaling after tamoxifen treatment, MCF-7 cells were treated with IGF-1 in conditioned medium (CM) obtained from 4-OHT-treated MCF-7 cells and the accumulation of phospho-Akt (S473) was measured. CM inhibited IGF-1-dependent cell signaling and suggesting the involvement of extracellular factors (ie. IGFBPs). A significant increase in IGFBP-1 mRNA and extracellular IGFBP-1 protein was observed in 4-OHT-treated MCF-7 cells. Knockdown experiments demonstrated that both GPER1 and CREB mediate IGFBP-1 induction. Furthermore, experiments showed that 4-OHT-dependent IGFBP-1 transcription is downstream of GPER1-activation in breast cancer cells. Additionally, neutralization and knockdown experiments demonstrated a role for IGFBP-1 in the observed inhibition of IGF-1 signaling. These results suggested that 4-OHT inhibits IGF-1 signaling via GPER1 and CREB mediated extracellular IGFBP-1 accumulation in breast cancer cells. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  6. Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

    2013-10-11

    Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

  7. Adiponectin mediated MHC class II mismatched cardiac graft rejection in mice is IL-4 dependent.

    Directory of Open Access Journals (Sweden)

    Daxu Li

    Full Text Available BACKGROUND: Adiponectin regulates glucose and fatty-acid metabolism but its role in chronic graft rejection mediated by Th2 cytokines remains ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: Wild type and adiponectin-null mice were used as graft recipients in mouse MHC class II disparate cardiac transplantation (bm12 toB6 and the graft rejection was monitored. In adiponectin-null mice we observed that the cellular infiltrate of eosinophils, CD4(+ and CD8(+ T cells was reduced in grafts compared to the controls as was collagen deposition and vessel occlusion. A similar outcome was observed for skin transplants except that neutrophil infiltration was increased. Low levels of IL-4 were detected in the grafts and serum. The effect of adiponectin signaling on IL-4 expression was further investigated. Treatment with AMPK and p38 MAPK inhibitors blocked adiponectin enhanced T cell proliferation in mixed lymphocyte reactions. Inhibition of AMPK reduced eosinophil infiltration in skin grafts in wild type recipients and in contrast AMPK activation increased eosinophils in adiponectin-null recipients. The addition of adiponectin increased IL-4 production by the T cell line EL4 with augmented nuclear GATA-3 and phospho-STAT6 expression which were suppressed by knockdown of adiponectin receptor 1 and 2. CONCLUSIONS: Our results demonstrate a direct effect of adiponectin on IL-4 expression which contributes to Th2 cytokine mediated rejection in mouse MHC class II histoincompatible transplants. These results add to our understanding of the interrelationship of metabolism and immune regulation and raise the possibility that AMPK inhibitors may be beneficial in selected types of rejection.

  8. Monocyte-mediated Serum-independent Damage to Hyphal and Pseudohyphal Forms of Candida albicans In Vitro

    OpenAIRE

    Diamond, Richard D.; Haudenschild, Christian C.

    1981-01-01

    Human peripheral blood monocytes attached to Candida albicans hyphae in the absence of serum and damaged the hyphae without completely ingesting them. Attachment and damage was not augmented by the addition of serum. Damage to hyphae was quantitated by a previously developed metabolic assay that measured leukocyte-induced reduction in uptake of [14C]cytosine by the hyphae. Use of cells from patients with hereditary disorders of leukocyte function, chronic granulomatous disease, and myeloperox...

  9. mediation: R Package for Causal Mediation Analysis

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    Dustin Tingley

    2014-09-01

    Full Text Available In this paper, we describe the R package mediation for conducting causal mediation analysis in applied empirical research. In many scientific disciplines, the goal of researchers is not only estimating causal effects of a treatment but also understanding the process in which the treatment causally affects the outcome. Causal mediation analysis is frequently used to assess potential causal mechanisms. The mediation package implements a comprehensive suite of statistical tools for conducting such an analysis. The package is organized into two distinct approaches. Using the model-based approach, researchers can estimate causal mediation effects and conduct sensitivity analysis under the standard research design. Furthermore, the design-based approach provides several analysis tools that are applicable under different experimental designs. This approach requires weaker assumptions than the model-based approach. We also implement a statistical method for dealing with multiple (causally dependent mediators, which are often encountered in practice. Finally, the package also offers a methodology for assessing causal mediation in the presence of treatment noncompliance, a common problem in randomized trials.

  10. Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

    Science.gov (United States)

    Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

    Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

  11. Radioimmunoassay of haloperidol in human serum: correlation of serum haloperidol with serum prolactin

    International Nuclear Information System (INIS)

    Poland, R.E.; Rubin, R.T.

    1981-01-01

    A radioimmunoassay (RIA) for measurement of serum haloperidol is described. Compared to gaschromatography (GC), RIA vaues average 40% higher. However, a simple organic extraction of serum yields statistically equivalent RIA and GC haloperidol determinations. For both men and women combined, there was a positive correlation between dose (mg/kg/day) and steady-state serum haloperidol level (r = +0.86) and between steady-state serum haloperidol and serum prolactin (PRL) concentration

  12. Progesterone Withdrawal-Evoked Plasticity of Neural Function in the Female Periaqueductal Grey Matter

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    T. A. Lovick

    2009-01-01

    Full Text Available Cyclical changes in production of neuroactive steroids during the oestrous cycle induce significant changes in GABAA receptor expression in female rats. In the periaqueductal grey (PAG matter, upregulation of α4β1δ GABAA receptors occurs as progesterone levels fall during late dioestrus (LD or during withdrawal from an exogenous progesterone dosing regime. The new receptors are likely to be extrasynaptically located on the GABAergic interneurone population and to mediate tonic currents. Electrophysiological studies showed that when α4β1δ GABAA receptor expression was increased, the excitability of the output neurones in the PAG increased, due to a decrease in the level of ongoing inhibitory tone from the GABAergic interneurones. The functional consequences in terms of nociceptive processing were investigated in conscious rats. Baseline tail flick latencies were similar in all rats. However, acute exposure to mild vibration stress evoked hyperalgesia in rats in LD and after progesterone withdrawal, in line with the upregulation of α4β1δ GABAA receptor expression.

  13. Interventional Effects for Mediation Analysis with Multiple Mediators.

    Science.gov (United States)

    Vansteelandt, Stijn; Daniel, Rhian M

    2017-03-01

    The mediation formula for the identification of natural (in)direct effects has facilitated mediation analyses that better respect the nature of the data, with greater consideration of the need for confounding control. The default assumptions on which it relies are strong, however. In particular, they are known to be violated when confounders of the mediator-outcome association are affected by the exposure. This complicates extensions of counterfactual-based mediation analysis to settings that involve repeatedly measured mediators, or multiple correlated mediators. VanderWeele, Vansteelandt, and Robins introduced so-called interventional (in)direct effects. These can be identified under much weaker conditions than natural (in)direct effects, but have the drawback of not adding up to the total effect. In this article, we adapt their proposal to achieve an exact decomposition of the total effect, and extend it to the multiple mediator setting. Interestingly, the proposed effects capture the path-specific effects of an exposure on an outcome that are mediated by distinct mediators, even when-as often-the structural dependence between the multiple mediators is unknown, for instance, when the direction of the causal effects between the mediators is unknown, or there may be unmeasured common causes of the mediators.

  14. Preliminary effects of pagoclone, a partial GABAA agonist, on neuropsychological performance

    Directory of Open Access Journals (Sweden)

    Angela F Caveney

    2008-03-01

    Full Text Available Angela F Caveney1, Bruno Giordani1, George M Haig21Department of Psychiatry, University of Michigan, Ann Arbor, MI, USA; 2Neurosciences Development, Abbott Laboratories, Abbott Park, IL, USAAbstract: Pagoclone is a novel cyclopyrrolone that acts as a partial GABAA receptor agonist. Preclinical studies suggest that pagoclone may have clinical utility as an anxiolytic agent, as well as a reduced incidence of side-effects. The present study was conducted to determine whether pagoclone would affect healthy individuals’ performances on neuropsychological measures as a function of dose within the projected therapeutic range. Twelve healthy adult subjects were randomly assigned to dosage groups in a 3-way crossover study. Participants were administered neuropsychological measures six hours following dosing on Day 1 and Day 6 of administration of the drug. Dose effects were noted on measures of alertness, learning, and memory and movement time. Significant effects were also noted on measures of alertness, learning and memory, information processing and psychomotor speed. Overall, the results of this small, preliminary study do not support a finding of behavioral toxicity for these doses of pagoclone. Rather, a pattern was found of transient and mild negative effects on learning and memory scores at the highest dose administered, though these changes were small and no longer evident by the sixth day of use.Keywords: pagoclone, cyclopyrrolone, neuropsychological, memory, generalized anxiety disorder

  15. Pioneer round of translation occurs during serum starvation

    International Nuclear Information System (INIS)

    Oh, Nara; Kim, Kyoung Mi; Cho, Hana; Choe, Junho; Kim, Yoon Ki

    2007-01-01

    The pioneer round of translation plays a role in translation initiation of newly spliced and exon junction complex (EJC)-bound mRNAs. Nuclear cap-binding protein complex CBP80/20 binds to those mRNAs at the 5'-end, recruiting translation initiation complex. As a consequence of the pioneer round of translation, the bound EJCs are dissociated from mRNAs and CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E. Steady-state translation directed by eIF4E allows for an immediate and rapid response to changes in physiological conditions. Here, we show that nonsense-mediated mRNA decay (NMD), which restricts only to the pioneer round of translation but not to steady-state translation, efficiently occurs even during serum starvation, in which steady-state translation is drastically abolished. Accordingly, CBP80 remains in the nucleus and processing bodies are unaffected in their abundance and number in serum-starved conditions. These results suggest that mRNAs enter the pioneer round of translation during serum starvation and are targeted for NMD if they contain premature termination codons

  16. Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions.

    Science.gov (United States)

    Patrikoski, Mimmi; Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

    2017-01-01

    Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

  17. The serum concentration of copper in bipolar disorder.

    Science.gov (United States)

    Siwek, Marcin; Styczeń, Krzysztof; Sowa-Kućma, Magdalena; Dudek, Dominika; Reczyński, Witold; Szewczyk, Bernadeta; Misztak, Paulina; Opoka, Włodzimierz; Topór-Mądry, Roman; Nowak, Gabriel; Rybakowski, Janusz K

    2017-06-18

    Some scientific reports indicate the changes in the concentration of serum copper in patients with bipolar disorder (BD), however the data are inconclusive. The aim of this study was to assess the concentration of copper in the blood serum of patients in various phases of BD compared to healthy volunteers, taking into consideration the specific clinical features, and the stage of illness. The study enrolled 133 patients with a diagnosis of BD (type I, II and NOS), including 61 people in depressive episode, 23 in mania or hypomania and 49 in remission. The control group consisted of 50 people. Atomic absorption spectrometry was used to measure the concentration of copper. There were no statistically significant differences in the serum copper concentration between patients in various phases of BD (mania/hypomania, depression, remission), sub-types (Type I, Type II + NOS) or stages and healthy volunteers. However, serum copper concentrations in patients in stage 1 was significantly higher than in advanced stages (2+3+4), (ß = 0.22; p = 0.02). Serum copper concentration was also the higher, the later the age of onset was (ß = 0.33; p < 0.001), and the lower, the greater the number of illness episodes (ß = - 0.23; p = 0.02) (multiple regression model, adj R2 = 0.19, p = 0.0001). The dependencies demonstrated above may reflect pathophysiological processes that occur in the course of BD (e.g., inflammatory response and oxidative stress) with a different intensity depending on its stage.

  18. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Mucorales species activation of a serum leukotactic factor.

    Science.gov (United States)

    Marx, R S; Forsyth, K R; Hentz, S K

    1982-01-01

    Previous studies have suggested that the focal accumulation of phagocytic leukocytes is an important feature of the host response in mucormycosis. To ascertain the basis for this influx of inflammatory cells, we evaluated the effect of members of the order Mucorales, including species from the genera Rhizopus, Absidia, and Mucor, on the chemotactic activity of normal human serum for neutrophils and monocytes. Both hyphae and spores produced concentration-dependent chemotaxigenesis in serum to a maximum level equivalent to that produced by zymosan activation of serum. Chemotactic activity was similar for live and heat-killed hyphae. No leukotactic activity was demonstrated in the absence of serum. The pretreatment of serum with anti-C3 antibody, heating at 56 degrees C, or 0.01 M EDTA abolished the activity. The pretreatment of serum with 0.01 M ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid did not abolish the activity. These data provide evidence that the leukotactic activity of Mucorales species is generated through the alternative complement pathway. PMID:6759409

  20. A cohort effect on serum testosterone levels in Finnish men

    DEFF Research Database (Denmark)

    Perheentupa, A; Mäkinen, J; Laatikainen, T

    2013-01-01

    To investigate whether a population-level decline in serum testosterone exists in Finnish men. In comparison with other European populations, Finnish men have compared well in the studies of reproductive health (i.e. semen quality, incidence of cryptorchidism and testicular cancer); thus, we...... expected no significant cohort-dependent decrease in serum testosterone....

  1. The value of serum procalcitonin content for assessing the inflammation and organ injury in neonatal septicemia

    Directory of Open Access Journals (Sweden)

    Shao-Fang Li1

    2017-04-01

    Full Text Available Objective: To study the value of serum procalcitonin content for assessing the inflammation and organ injury in neonatal septicemia. Methods: 48 children with neonatal septicemia who were treated in our hospital between April 2014 and May 2016 were selected as the observation group, 50 healthy newborns who were delivered in our hospital during the same period were selected as the normal control group, and the observation group were further divided into high PCT group and low PCT group (n=24 according to the median of serum PCT content. Enzyme-linked immunosorbent assay (ELISA was used to detect the serum contents of inflammatory mediators, and color Doppler diasonograph was used to measure heart injury index levels. Results: Peripheral blood PCT content of observation group was significantly higher than that of control group (P<0.05; serum inflammatory mediators IL-1β, IL-6, IL-8 and TNF-α contents of high PCT group and low PCT group were significantly higher than those of normal control group, and as the PCT content increased, serum inflammatory mediators interleukin-1β (IL-1β, interleukin-6 (IL-6, interleukin-8 (IL-8 and tumor necrosis factor-α (TNF-α contents increased (P<0.05; routine ultrasound parameters cardiac output (CO and left ventricular ejection fraction (LVEF levels as well as the absolute value of twodimensional speckle tracking imaging parameters left ventricular global longitudinal strain rate (GLSr and left ventricular global circumferential strain rate (GCSr of high PCT group and low PCT group were lower than those of normal control group while serum myocardial injury indexes cardiac troponin I (cTnI, heart-type fatty acid-binding protein (H-FABP and α-hydroxybutyric dehydrogenase (HBDH contents were higher than those of normal control group, and as the PCT content increased, CO and LVEF levels as well as the absolute value of GLSr and GCSr decreased while the indexes cTnI, H-FABP and HBDH contents increased (P<0

  2. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Science.gov (United States)

    Zhang, Yu-Qin; Xiao, Chuan-Xing; Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  3. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Yu-Qin Zhang

    Full Text Available The role of Pokemon (POK erythroid myeloid ontogenic actor, a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  4. Association Between Smoking, Nicotine Dependence, and BDNF Val(66)Met Polymorphism with BDNF Concentrations in Serum

    NARCIS (Netherlands)

    Jamal, Mumtaz; Van der Does, Willem; Elzinga, Bernet M.; Molendijk, Marc L.; Penninx, Brenda W. J. H.

    Introduction: Nicotine use is associated with the upregulation of brain-derived neurotrophic factor (BDNF) in serum. An association between smoking and the BDNF Val(66)Met polymorphism has also been found. The aim of this study is to examine the levels of serum BDNF in never-smokers, former smokers,

  5. Investigation of interactions between dendrimer-coated magnetite nanoparticles and bovine serum albumin

    International Nuclear Information System (INIS)

    Pan Bifeng; Gao Feng; Ao Limei

    2005-01-01

    We investigated the interactions between dendrimer-coated magnetite nanoparticles (MNPs) and the protein serum albumin. The investigation was based on the fluorescence quenching of tryptophan residue of serum albumin after binding with the dendrimer-coated magnetite nanoparticles. The extent of the interactions between bovine serum albumin and dendrimer-coated MNPs strongly depends on their surface groups and pH value

  6. Radioimmunological determination of digoxin in serum

    International Nuclear Information System (INIS)

    Gruber, G.; Sorger, D.

    1979-01-01

    The tested quality signs of the digoxin-RIA (Medica) correspond to other RIA-test methods. The digoxin-RIA (Medica) is, therefore, well suited for clinical examinations. In the dilanacin-(digoxin-) long-term therapy with the maintenance dose of 0.5 mg digoxin a day 75.4% of the patients examined (n = 65) were within the therapeutic field. In each case 12.3% were underdigitalised or overdigitalised, respectively. In the 8 patients in the toxic region only the half showed signs of digoxin intoxication. It is referred to the importance of creatinine and potassium in the serum, to the digoxin creatinene and potassium in the serum, to the digoxin clearance depending on age, to the body weight and the body surface, respectively, in very obese patients for the height of the serum digoxin level and for the compatibility of this heart glycoside. (author)

  7. Dependency, self-criticism and negative affective responses following imaginary rejection and failure threats: meaning-making processes as moderators or mediators.

    Science.gov (United States)

    Besser, Avi; Priel, Beatriz

    2011-01-01

    This study evaluated the intervening role of meaning-making processes in emotional responses to negative life events based on Blatt's (1974, 2004) formulations concerning the role of personality predispositions in depression. In a pre/post within-subject study design, a community sample of 233 participants reacted to imaginary scenarios of interpersonal rejection and achievement failure. Meaning-making processes relating to threats to self-definition and interpersonal relatedness were examined following the exposure to the scenarios. The results indicated that the personality predisposition of Dependency, but not Self-Criticism predicted higher levels of negative affect following the interpersonal rejection event, independent of baseline levels of negative affect. This effect was mediated by higher levels of negative meaning-making processes related to the effect of the interpersonal rejection scenario on Dependent individuals' senses of interpersonal relatedness and self-worth. In addition, both Self-Criticism and Dependency predicted higher levels of negative affect following the achievement failure event, independent of baseline levels of negative affect. Finally, the effect of Self-Criticism was mediated by higher levels of negative meaning-making processes related to the effect of the achievement failure scenario on self-critical individuals' senses of self-definition.

  8. Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression in U937 cells.

    Science.gov (United States)

    Chihara, Kazuyasu; Kato, Yuji; Yoshiki, Hatsumi; Takeuchi, Kenji; Fujieda, Shigeharu; Sada, Kiyonao

    2017-09-13

    The adaptor protein c-Abl SH3 domain binding protein-2 (3BP2) is tyrosine phosphorylated by Syk in response to cross-linking of antigen receptors, which in turn activates various immune responses. Recently, a study using the mouse model of cherubism, a dominant inherited disorder caused by mutations in the gene encoding 3BP2, showed that 3BP2 is involved in the regulation of phagocytosis mediated by Fc receptor for IgG (FcγR) in macrophages. However, the molecular mechanisms underlying 3BP2-mediated regulation of phagocytosis and the physiological relevance of 3BP2 tyrosine phosphorylation remains elusive. In this study, we established various gene knockout U937 cell lines using the CRISPR/Cas9 system and found that 3BP2 is rapidly tyrosine phosphorylated by Syk in response to cross-linking of FcγRI. Depletion of 3BP2 caused significant reduction in the Fc receptor γ chain (FcRγ)-mediated phagocytosis in addition to the FcγRI-mediated induction of chemokine mRNA for IL-8, CCL3L3 and CCL4L2. Syk-dependent tyrosine phosphorylation of 3BP2 was required for overcoming these defects. Finally, we found that the PH and SH2 domains play important roles on FcγRI-mediated tyrosine phosphorylation of 3BP2 in HL-60 cells. Taken together, these results indicate that Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression.

  9. Lack of Radiation Dose or Quality Dependence of Epithelial-to-Mesenchymal Transition (EMT) Mediated by Transforming Growth Factor β

    International Nuclear Information System (INIS)

    Andarawewa, Kumari L.; Costes, Sylvain V.; Fernandez-Garcia, Ignacio; Chou, William S.; Ravani, Shraddha A.; Park, Howard; Barcellos-Hoff, Mary Helen

    2011-01-01

    Purpose: Epithelial-to-mesenchymal transition (EMT) is a phenotype that alters cell morphology, disrupts morphogenesis, and increases motility. Our prior studies have shown that the progeny of human mammary epithelial cells (HMECs) irradiated with 2 Gy undergoes transforming growth factor β (TGF-β)-mediated EMT. In this study we determined whether radiation dose or quality affected TGF-β-mediated EMT. Methods and Materials: HMECs were cultured on tissue culture plastic or in Matrigel (BD Biosciences, San Jose, CA) and exposed to low or high linear energy transfer (LET) and TGF-β (400 pg/mL). Image analysis was used to measure membrane-associated E-cadherin, a marker of functional epithelia, or fibronectin, a product of mesenchymal cells, as a function of radiation dose and quality. Results: E-cadherin was reduced in TGF-β-treated cells irradiated with low-LET radiation doses between 0.03 and 2 Gy compared with untreated, unirradiated cells or TGF-β treatment alone. The radiation quality dependence of TGF-β-mediated EMT was determined by use of 1 GeV/amu (gigaelectron volt / atomic mass unit) 56 Fe ion particles at the National Aeronautics and Space Administration's Space Radiation Laboratory. On the basis of the relative biological effectiveness of 2 for 56 Fe ion particles' clonogenic survival, TGF-β-treated HMECs were irradiated with equitoxic 1-Gy 56 Fe ion or 2-Gy 137 Cs radiation in monolayer. Furthermore, TGF-β-treated HMECs irradiated with either high- or low-LET radiation exhibited similar loss of E-cadherin and gain of fibronectin and resulted in similar large, poorly organized colonies when embedded in Matrigel. Moreover, the progeny of HMECs exposed to different fluences of 56 Fe ion underwent TGF-β-mediated EMT even when only one-third of the cells were directly traversed by the particle. Conclusions: Thus TGF-β-mediated EMT, like other non-targeted radiation effects, is neither radiation dose nor quality dependent at the doses examined.

  10. Diselenolane-mediated cellular uptake.

    Science.gov (United States)

    Chuard, Nicolas; Poblador-Bahamonde, Amalia I; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi; Matile, Stefan

    2018-02-21

    The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides.

  11. Malaria-induced NLRP12/NLRP3-dependent caspase-1 activation mediates inflammation and hypersensitivity to bacterial superinfection.

    Directory of Open Access Journals (Sweden)

    Marco A Ataide

    2014-01-01

    Full Text Available Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14(+CD16(-Caspase-1(+ and CD14(dimCD16(+Caspase-1(+ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria.

  12. Propofol reduced myocardial contraction of vertebrates partly by mediating the cyclic AMP-dependent protein kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Sun, Xiaotong; Zhang, Xinyu; Bo, Qiyu; Meng, Tao; Lei, Zhen; Li, Jingxin; Hou, Yonghao; Yu, Xiaoqian; Yu, Jingui

    2016-01-01

    Propofol inhibits myocardial contraction in a dose dependent manner. The present study is designed to examine the effect of propofol on PKA mediated myocardial contraction in the absence of adrenoreceptor agonist. The contraction of isolated rat heart was measured in the presence or absence of PKA inhibitor H89 or propofol, using a pressure transducer. The levels of cAMP and PKA kinase activity were detected by ELISA. The mRNA and total protein or phosphorylation level of PKA and downstream proteins were tested in the presence or absence of PKA inhibitor H89 or propofol, using RT-PCR, QPCR and western blotting. The phosphorylation level of PKA was examined thoroughly using immunofluorescence and PKA activity non-radioactive detection kit. Propofol induced a dose-dependent negative contractile response on the rat heart. The inhibitory effect of high concentration propofol (50 μM) with 45% decease of control could be partly reversed by the PKA inhibitor H89 (10 μM) and the depressant effect of propofol decreased from 45% to 10%. PKA kinase activity was inhibited by propofol in a dose-dependent manner. Propofol also induced a decrease in phosphorylation of PKA, which was also inhibited by H89, but did not alter the production of cAMP and the mRNA levels of PKA. The downstream proteins of PKA, PLN and RyR2 were phosphorylated to a lesser extent with propofol or H89 than control. These results demonstrated that propofol induced a negative myocardial contractile response partly by mediating the PKA phosphorylation pathway.

  13. NMDAR-mediated calcium transients elicited by glutamate co-release at developing inhibitory synapses

    Directory of Open Access Journals (Sweden)

    Abigail Kalmbach

    2010-07-01

    Full Text Available Before hearing onset, the topographic organization of the inhibitory sound localization pathway from the medial nucleus of the trapezoid body (MNTB to the lateral superior olive (LSO is refined by means of synaptic silencing and strengthening. During this refinement period MNTB-LSO synapses not only release GABA and glycine but also release glutamate. This co-released glutamate can elicit postsynaptic currents that are predominantly mediated by NMDA receptors (NMDARs. To gain a better understanding of how glutamate contributes to synaptic signaling at developing MNTB-LSO inhibitory synapse, we investigated to what degree and under what conditions NMDARs contribute to postsynaptic calcium responses. Our results demonstrate that MNTB-LSO synapses can elicit compartmentalized calcium responses along aspiny LSO dendrites. These responses are significantly attenuated by the NMDARs antagonist APV. APV, however, has no effect on somatically recorded electrical postsynaptic responses, indicating little, if any, contribution of NMDARs to spike generation. Small NMDAR-mediated calcium responses were also observed under physiological levels of extracellular magnesium concentrations indicating that MNTB-LSO synapses activate magnesium sensitive NMDAR on immature LSO dendrites. In Fura-2 AM loaded neurons, blocking GABAA and glycine receptors decreased NMDAR contribution to somatic calcium responses suggesting that GABA and glycine, perhaps by shunting backpropagating action potentials, decrease the level of NMDAR activation under strong stimulus conditions.

  14. A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

    Directory of Open Access Journals (Sweden)

    Eleonora Dehlink

    Full Text Available Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI, the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

  15. Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells.

    Science.gov (United States)

    Park, Jungyun; Ahn, Seyoung; Jayabalan, Aravinth K; Ohn, Takbum; Koh, Hyun Chul; Hwang, Jungwook

    2016-07-01

    Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to impair NMD; however, few studies have investigated the correlation between enhanced translation and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound NMD targets. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Angiotensin II and CRF receptors in the central nucleus of the amygdala mediate hemodynamic response variability to cocaine in conscious rats.

    Science.gov (United States)

    Watanabe, Mari A; Kucenas, Sarah; Bowman, Tamara A; Ruhlman, Melissa; Knuepfer, Mark M

    2010-01-14

    Stress or cocaine evokes either a large increase in systemic vascular resistance (SVR) or a smaller increase in SVR accompanied by an increase in cardiac output (designated vascular and mixed responders, respectively) in Sprague-Dawley rats. We hypothesized that the central nucleus of the amygdala (CeA) mediates this variability. Conscious, freely-moving rats, instrumented for measurement of arterial pressure and cardiac output and for drug delivery into the CeA, were given cocaine (5 mg/kg, iv, 4-6 times) and characterized as vascular (n=15) or mixed responders (n=10). Subsequently, we administered cocaine after bilateral microinjections (100 nl) of saline or selective agents in the CeA. Muscimol (80 pmol), a GABA(A) agonist, or losartan (43.4 pmol), an AT(1) receptor antagonist, attenuated the cocaine-induced increase in SVR in vascular responders, selectively, such that vascular responders were no longer different from mixed responders. The corticotropin releasing factor (CRF) antagonist, alpha-helical CRF(9-41) (15.7 pmol), abolished the difference between cardiac output and SVR in mixed and vascular responders. We conclude that greater increases in SVR observed in vascular responders are dependent on AT(1) receptor activation and, to a lesser extent on CRF receptors. Therefore, AT(1) and CRF receptors in the CeA contribute to hemodynamic response variability to intravenous cocaine.

  17. Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

    Science.gov (United States)

    Citrus paradisi Macf. cv. Duncan was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from CTV genome and used to gener...

  18. Serum C-reactive protein concentrations in Nova Scotia Duck Tolling Retrievers with immune-mediated rheumatic disease.

    Science.gov (United States)

    Bremer, Hanna Dorotea; Hillström, Anna; Kånåhols, Malin; Hagman, Ragnvi; Hansson-Hamlin, Helene

    2017-04-17

    Nova Scotia Duck Tolling Retrievers (NSDTRs) are a dog breed often affected by immune-mediated rheumatic disease (IMRD), a disorder characterised by chronic stiffness and joint pain. Most, but not all, dogs with IMRD, have antinuclear antibodies (ANA), which are also commonly present in the autoimmune disease systemic lupus erythematosus (SLE). The clinical and diagnostic findings of IMRD indicate that it is an SLE-related disorder. C-reactive protein (CRP), an acute phase protein, is a quantitative marker of inflammation for many diseases and is used for diagnosing and monitoring systemic inflammation in both humans and dogs. However, in human SLE, CRP concentrations are often elevated but correlate poorly with disease activity; they can be low in individual patients with active disease. The aim of the study was to investigate CRP in a group of NSDTRs with the SLE-related disorder IMRD. The hypothesis was that CRP concentrations would be increased in dogs with IMRD compared to healthy dogs, but that the increase would be mild. Serum CRP concentrations were measured in 18 IMRD-affected NSDTRs and 19 healthy control NSDTRs using two different canine-specific CRP assays. Dogs with IMRD and ANA had higher CRP concentrations than the control dogs, but the concentrations were below the clinical decision limit for systemic inflammation for most of the IMRD dogs. These results indicate that CRP concentrations were increased in dogs with IMRD and ANA, but the increase was mild, similar to what has been observed in human SLE.

  19. Shear rate normalization is not essential for removing the dependency of flow-mediated dilation on baseline artery diameter: past research revisited

    International Nuclear Information System (INIS)

    Atkinson, Greg

    2014-01-01

    A ratio index (FMD%) is used ubiquitously to scale (by simple division) brachial artery flow-mediated dilation (D diff ) in direct proportion to baseline diameter (D base ). It is now known that D diff is inversely proportional to D base rendering FMD% wholly inappropriate. Consequently, FMD% is still substantially dependent on D base . Although this problem is grounded in statistics, normalization of FMD% for the change in arterial shear rate (ΔSR) has been proposed to remove this D base -dependency. It was hypothesized that, if the flow-mediated response is scaled properly to D base in the first place, shear rate normalization would not be needed to remove D base -dependency. Dedicated software (Digitizelt) was employed to extract the data from a seminal study on FMD% normalization. The underlying allometric relationship between D base and peak diameter (D peak ) was described. The re-analyses revealed that the absolute change in arterial diameter was strongly inversely proportional to D base (r= − 0.7, P < 0.0005). The allometric exponent for the D base –D peak relationship was 0.82 (95% CI: 0.78–0.86) rather than the value of 1 needed for appropriate use of FMD%. The allometric approach completely eliminated the originally reported dependency on D base without any need for ΔSR normalization (r=0.0, P=0.96). The correlation between ΔSR and FMD% reduced from 0.69 to 0.37, when adjusted for D base . In conclusion, this new re-analysis of data from an influential study demonstrates that the FMD%–D base correlation is caused by the inappropriate size-scaling properties of FMD% itself. Removal of D base -dependency via FMD%/ΔSR normalization is not essential at all if allometric scaling is applied to isolate the flow-mediated response in the first place. Consequently, the influence of ΔSR on this properly scaled response can also be isolated and quantified accurately without the confounding influence of D base . (paper)

  20. Low serum zinc is associated with elevated risk of cadmium nephrotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yu-Sheng, E-mail: Lin.Yu-Sheng@epa.gov [National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC (United States); Ho, Wen-Chao [Department of Public Health, College of Public Health, China Medical University, Taichung, Taiwan (China); Caffrey, James L. [Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, TX (United States); Sonawane, Babasaheb [National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC (United States)

    2014-10-15

    Background: Despite animal evidence suggests that zinc modulates cadmium nephrotoxicity, limited human data are available. Objective: To test the hypothesis that low serum zinc concentrations may increase the risk of cadmium-mediated renal dysfunction in humans. Methods: Data from 1545 subjects aged 20 or older in the National Health and Nutrition Examination Survey (NHANES), 2011–2012 were analyzed. Renal function was defined as impaired when estimated glomerular filtration rate (eGFR) fell below 60 ml/min/1.73 m{sup 2} and/or the urinary albumin-to-creatinine ratio surpassed 2.5 in men and 3.5 mg/mmol in women. Results: Within the study cohort, 117 subjects had reduced eGFR and 214 had elevated urinary albumin. After adjusting for potential confounders, subjects with elevated blood cadmium (>0.53 μg/L) were more likely to have a reduced eGFR (odds ratio [OR]=2.21, 95% confidence interval [CI]: 1.09–4.50) and a higher urinary albumin (OR=2.04, 95% CI: 1.13–3.69) than their low cadmium (<0.18 μg/L) peers. In addition, for any given cadmium exposure, low serum zinc is associated with elevated risk of reduced eGFR (OR=3.38, 95% CI: 1.39–8.28). A similar increase in the odds ratio was observed between declining serum zinc and albuminuria but failed to reach statistical significance. Those with lower serum zinc/blood cadmium ratios were likewise at a greater risk of renal dysfunction (p<0.01). Conclusions: This study results suggest that low serum zinc concentrations are associated with an increased risk of cadmium nephrotoxicity. Elevated cadmium exposure is global public health issue and the assessment of zinc nutritional status may be an important covariate in determining its effective renal toxicity. - Highlights: • Blood cadmium was associated with increased risk of nephrotoxicity. • Low serum zinc may exacerbate risk of cadmium-mediated renal dysfunction. • Both zinc deficiency and elevated cadmium exposure are global public health issues.

  1. Serum thyroglobulin as a biomarker of iodine deficiency in adult populations

    DEFF Research Database (Denmark)

    Krejbjerg, Anne; Bjergved, Lena; Bülow Pedersen, Inge

    2016-01-01

    OBJECTIVE: To clarify which factors may influence the serum Tg level in an adult population and how this may affect Tg as a biomarker of iodine deficiency (ID). DESIGN AND METHODS: Two identical cross-sectional studies were performed before (C1a: 1997-98, n = 4649) and after (C2: 2004-05, n = 3570......) the Danish mandatory iodine fortification (IF) of salt (2000). Additionally, a follow-up study of C1a was performed after IF (C1b: 2008-10, n = 2465). The studies took place in two regions with mild (Copenhagen) and moderate (Aalborg) ID before IF. Serum Tg was measured by immunoradiometric method...... and investigated as outcome variable in multivariate models. RESULTS: Multiple factors were associated with serum Tg. Some were directly related to iodine intake (cohort, urinary iodine concentration (UIC) level and region), and some were likely mediators of iodine intake effects on Tg (thyroid nodularity, thyroid...

  2. PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1.

    Science.gov (United States)

    Iida, Satoshi; Chen, Wei; Nakadai, Tomoyoshi; Ohkuma, Yoshiaki; Roeder, Robert G

    2015-02-01

    PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex. © 2015 Iida et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

    Science.gov (United States)

    Beltrán González, Andrea N; Vicentini, Florencia; Calvo, Daniel J

    2018-01-01

    l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABA A ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABA A ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABA A ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABA A ρ1 receptors. © 2017 International Society for Neurochemistry.

  4. Current Directions in Mediation Analysis

    Science.gov (United States)

    MacKinnon, David P.; Fairchild, Amanda J.

    2010-01-01

    Mediating variables continue to play an important role in psychological theory and research. A mediating variable transmits the effect of an antecedent variable on to a dependent variable, thereby providing more detailed understanding of relations among variables. Methods to assess mediation have been an active area of research for the last two decades. This paper describes the current state of methods to investigate mediating variables. PMID:20157637

  5. Current Directions in Mediation Analysis

    OpenAIRE

    MacKinnon, David P.; Fairchild, Amanda J.

    2009-01-01

    Mediating variables continue to play an important role in psychological theory and research. A mediating variable transmits the effect of an antecedent variable on to a dependent variable, thereby providing more detailed understanding of relations among variables. Methods to assess mediation have been an active area of research for the last two decades. This paper describes the current state of methods to investigate mediating variables.

  6. Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway.

    Science.gov (United States)

    Leopold, Philip L; Wendland, Rebecca L; Vincent, Theresa; Crystal, Ronald G

    2006-10-01

    Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non

  7. Characterization of Serum Phospholipase A2 Activity in Three Diverse Species of West African Crocodiles

    Directory of Open Access Journals (Sweden)

    Mark Merchant

    2011-01-01

    Full Text Available Secretory phospholipase A2, an enzyme that exhibits substantial immunological activity, was measured in the serum of three species of diverse West African crocodiles. Incubation of different volumes of crocodile serum with bacteria labeled with a fluorescent fatty acid in the sn-2 position of membrane lipids resulted in a volume-dependent liberation of fluorescent probe. Serum from the Nile crocodile (Crocodylus niloticus exhibited slightly higher activity than that of the slender-snouted crocodile (Mecistops cataphractus and the African dwarf crocodile (Osteolaemus tetraspis. Product formation was inhibited by BPB, a specific PLA2 inhibitor, confirming that the activity was a direct result of the presence of serum PLA2. Kinetic analysis showed that C. niloticus serum produced product more rapidly than M. cataphractus or O. tetraspis. Serum from all three species exhibited temperature-dependent PLA2 activities but with slightly different thermal profiles. All three crocodilian species showed high levels of activity against eight different species of bacteria.

  8. Heme oxygenase-1 regulates the progression of K/BxN serum transfer arthritis.

    Directory of Open Access Journals (Sweden)

    Rita Brines

    Full Text Available Heme oxygenase-1 (HO-1 is induced in many cell types as a defense mechanism against stress. We have investigated the possible role of endogenous HO-1 in the effector phase of arthritis using the K/BxN serum transfer model of arthritis in HO-1 heterozygous and homozygous knock-out mice.Arthritis was induced in C57/Black-6 xFVB (HO-1(+/+, HO-1(+/- and HO-1(-/- mice by intraperitoneal injection of 150 µl serum from arthritic K/BxN mice at days 0 and 2. Blood was collected and animals were sacrificed at day 10. Histological analysis was performed in ankle sections. The levels of inflammatory mediators were measured in serum and paw homogenates by enzyme-linked immunosorbent assay or Multiplex technology. The incidence of arthritis was higher in HO-1(+/- and HO-1(-/- groups compared with HO-1(+/+. The inflammatory response was aggravated in HO-1(+/- mice as shown by arthritic score and the migration of inflammatory cells that could be related to the enhancement of CXCL-1 production. In addition, the HO-1(+/- group showed proteoglycan depletion significantly higher than HO-1(+/+ mice. Serum levels of matrix metalloproteinase-3, monocyte chemotactic protein-1, plasminogen activator inhibitor-1, E-selectin and intercellular adhesion molecule-1 were increased in arthritic HO-1(-/- mice, whereas vascular endothelial growth factor and some cytokines such as interferon-γ showed a reduction compared to HO-1(+/+ or HO-1(+/- mice. In addition, down-regulated gene expression of ferritin, glutathione S-reductase A1 and superoxide dismutase-2 was observed in the livers of arthritic HO-1(+/- animals.Endogenous HO-1 regulates the production of systemic and local inflammatory mediators and plays a protective role in K/BxN serum transfer arthritis.

  9. Context-dependent modulation of alphabetagamma and alphabetadelta GABA A receptors by penicillin: implications for phasic and tonic inhibition.

    Science.gov (United States)

    Feng, Hua-Jun; Botzolakis, Emmanuel J; Macdonald, Robert L

    2009-01-01

    Penicillin, an open-channel blocker of GABA(A) receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABA(A) receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents evoked from both synaptic and extrasynaptic receptor isoforms. However, penicillin had isoform-specific effects on the extent of desensitization, reflecting its ability to differentially modulate peak (non-equilibrium) and residual (near-equilibrium) currents. This suggested that the context of activation could determine the apparent sensitivity of a given receptor isoform to penicillin. To test this hypothesis, we explored the ability of penicillin to modulate synaptic and extrasynaptic isoform currents that were activated under more physiologically relevant conditions. Interestingly, while currents evoked from synaptic isoforms under phasic conditions (transient activation by a saturating concentration of GABA) were substantially inhibited by penicillin, currents evoked from extrasynaptic isoforms under tonic conditions (prolonged application by a sub-saturating concentration of GABA) were minimally affected. We therefore concluded that the reported inability of penicillin to modulate tonic currents could not simply be attributed to insensitivity of extrasynaptic receptors, but rather, reflected an inability to modulate these receptors in their native context of activation.

  10. Mxi1 and Mxi1-0 Antagonize N-Myc Function and Independently Mediate Apoptosis in Neuroblastoma

    Directory of Open Access Journals (Sweden)

    David A. Erichsen

    2015-02-01

    Full Text Available Neuroblastoma (NB is the third most common malignancy of childhood, and outcomes for children with advanced disease remain poor; amplification of the MYCN gene portends a particularly poor prognosis. Mxi1 antagonizes N-Myc by competing for binding to Max and E-boxes. Unlike N-Myc, Mxi1 mediates transcriptional repression and suppresses cell proliferation. Mxi1 and Mxi1-0 (an alternatively transcribed Mxi1 isoform share identical Max and DNA binding domains but differ in amino-terminal sequences. Because of the conservation of these critical binding domains, we hypothesized that Mxi1-0 antagonizes N-Myc activity similar to Mxi1. SHEP NB cells and SHEP cells stably transfected with MYCN (SHEP/MYCN were transiently transfected with vectors containing full-length Mxi1, full-length Mxi1-0, or the common Mxi domain encoded by exons 2 to 6 (ex2-6. After incubation in low serum, parental SHEP/MYCN cell numbers were reduced compared with SHEP cells. Activated caspase-3 staining and DNA fragmentation ELISA confirmed that SHEP/MYCN cells undergo apoptosis in low serum, while SHEP/MYCN cells transfected with Mxi1 or Mxi1-0 do not. However, SHEP/MYCN cells transfected with Mxi1 or Mxi1-0 and grown in normal serum showed proliferation rates similar to SHEP cells. Mxi ex2-6 did not affect cell number in low or normal serum, suggesting that amino terminal domains of Mxi1 and Mxi1-0 are critical for antagonism. In the absence of N-Myc, Mxi1 and Mxi1-0 induce apoptosis independently through the caspase-8–dependent extrinsic pathway, while N-Myc activates the caspase-9–dependent intrinsic pathway. Together, these data indicate that Mxi1 and Mxi1-0 antagonize N-Myc but also independently impact NB cell survival.

  11. Role of glucocorticoid receptor-mediated mechanisms in cocaine memory enhancement.

    Science.gov (United States)

    Stringfield, S J; Higginbotham, J A; Wang, R; Berger, A L; McLaughlin, R J; Fuchs, R A

    2017-09-01

    The basolateral amygdala (BLA) is a critical site for the reconsolidation of labile contextual cocaine memories following retrieval-induced reactivation/destabilization. Here, we examined whether glucocorticoid receptors (GR), which are abundant in the BLA, mediate this phenomenon. Rats were trained to lever press for cocaine reinforcement in a distinct environmental context, followed by extinction training in a different context. Rats were then briefly exposed to the cocaine-paired context (to elicit memory reactivation and reconsolidation) or their home cages (no reactivation control). Exposure to the cocaine-paired context elicited greater serum corticosterone concentrations than home cage stay. Interestingly, the GR antagonist, mifepristone (3-10 ng/hemisphere), administered into the BLA after memory reactivation produced a further, dose-dependent increase in serum corticosterone concentrations during the putative time of cocaine-memory reconsolidation but produced an inverted U-shaped dose-effect curve on subsequent cocaine-seeking behavior 72 h later. This effect was anatomically selective, dependent on memory reactivation (i.e., not observed after home cage exposure), and did not reflect protracted hyperactivity. However, the effect was also observed when mifepristone was administered after novelty stress that mimics drug context-induced hypothalamic-pituitary-adrenal (HPA) axis activation without explicit memory reactivation. Together, these findings suggest that, similar to explicit memory retrieval, a stressful event is sufficient to destabilize cocaine memories and permit their manipulation. Furthermore, BLA GR stimulation exerts inhibitory feedback upon HPA axis activation and thus suppresses cocaine-memory reconsolidation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. A newly identified protein of Leptospira interrogans mediates binding to laminin.

    Science.gov (United States)

    Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2009-10-01

    Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

  13. Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

    Science.gov (United States)

    Lin, Chih-Chung; Lee, I-Ta; Hsu, Chun-Hao; Hsu, Chih-Kai; Chi, Pei-Ling; Hsiao, Li-Der; Yang, Chuen-Mao

    2015-01-01

    Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation. PMID:25734900

  14. Disinhibition, an emerging pharmacology of learning and memory [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Hanns Möhler

    2017-02-01

    Full Text Available Learning and memory are dependent on interactive excitatory and inhibitory mechanisms. In this review, we discuss a mechanism called disinhibition, which is the release of an inhibitory constraint that effectively results in an increased activity in the target neurons (for example, principal or projection neurons. We focus on discussing the role of disinhibition in learning and memory at a basic level and in disease models with cognitive deficits and highlight a strategy to reverse cognitive deficits caused by excess inhibition, through disinhibition of α5-containing GABAA receptors mediating tonic inhibition in the hippocampus, based on subtype-selective negative allosteric modulators as a novel class of drugs.

  15. Effects of serum on cytotoxicity of nano- and micro-sized ZnO particles

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, I-Lun; Huang, Yuh-Jeen, E-mail: yjhuang@mx.nthu.edu.tw [National Tsing Hua University, Department of Biomedical Engineering and Environmental Sciences (China)

    2013-09-15

    Although an increasing number of in vitro studies are being published regarding the cytotoxicity of nanomaterials, the components of the media for toxicity assays have often varied according to the needs of the scientists. Our aim for this study was to evaluate the influence of serum-in this case, fetal bovine serum-in a cell culture medium on the toxicity of nano-sized (50-70 nm) and micro-sized (<1 {mu}m) ZnO on human lung epithelial cells (A549). The nano- and micro-sized ZnO both exhibited their highest toxicity when exposed to serum-free media, in contrast to exposure in media containing 5 or 10 % serum. This mainly comes not only from the fact that ZnO particles in the serum-free media have a higher dosage-per-cell ratio, which results from large aggregates of particles, rapid sedimentation, absence of protein protection, and lower cell growth rate, but also that extracellular Zn{sup 2+} release contributes to cytotoxicity. Although more extracellular Zn{sup 2+} release was observed in serum-containing media, it did not contribute to nano-ZnO cytotoxicity. Furthermore, non-dissolved particles underwent size-dependent particle agglomeration, resulting in size-dependent toxicity in both serum-containing and serum-free media. A low correlation between cytotoxicity and inflammation endpoints in the serum-free medium suggested that some signaling pathways were changed or induced. Since cell growth, transcription behavior for protein production, and physicochemical properties of ZnO particles all were altered in serum-free media, we recommend the use of a serum-containing medium when evaluating the cytotoxicity of NPs.

  16. Blimp-1-Dependent IL-10 Production by Tr1 Cells Regulates TNF-Mediated Tissue Pathology.

    Directory of Open Access Journals (Sweden)

    Marcela Montes de Oca

    2016-01-01

    Full Text Available Tumor necrosis factor (TNF is critical for controlling many intracellular infections, but can also contribute to inflammation. It can promote the destruction of important cell populations and trigger dramatic tissue remodeling following establishment of chronic disease. Therefore, a better understanding of TNF regulation is needed to allow pathogen control without causing or exacerbating disease. IL-10 is an important regulatory cytokine with broad activities, including the suppression of inflammation. IL-10 is produced by different immune cells; however, its regulation and function appears to be cell-specific and context-dependent. Recently, IL-10 produced by Th1 (Tr1 cells was shown to protect host tissues from inflammation induced following infection. Here, we identify a novel pathway of TNF regulation by IL-10 from Tr1 cells during parasitic infection. We report elevated Blimp-1 mRNA levels in CD4+ T cells from visceral leishmaniasis (VL patients, and demonstrate IL-12 was essential for Blimp-1 expression and Tr1 cell development in experimental VL. Critically, we show Blimp-1-dependent IL-10 production by Tr1 cells prevents tissue damage caused by IFNγ-dependent TNF production. Therefore, we identify Blimp-1-dependent IL-10 produced by Tr1 cells as a key regulator of TNF-mediated pathology and identify Tr1 cells as potential therapeutic tools to control inflammation.

  17. Local impermeant anions establish the neuronal chloride concentration

    DEFF Research Database (Denmark)

    Glykys, J; Dzhala, V; Egawa, K

    2014-01-01

    Neuronal intracellular chloride concentration [Cl(-)](i) is an important determinant of γ-aminobutyric acid type A (GABA(A)) receptor (GABA(A)R)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl(-) across the membrane, but accumulat...

  18. Correlation between FSH, LH and prolactin serum levels. [Radioimmunoassay of hormones

    Energy Technology Data Exchange (ETDEWEB)

    Krause, W [Giessen Univ. (Germany, F.R.)

    1978-01-01

    In 188 males FSH, LH, and prolactin serum levels determined by radioimmunoassay from a single blood sample were found to be closely correlated. No correlation appeared to testosterone levels. The same correlation is observed, if serum levels of FSH, LH, and prolactin are measured after stimulation with LH-RH and TRH. In order to explain the close correlation, in five young men hormone levels were measured at 2-min-intervals over a period of 2 hours. Peaks of prolactin often correspond to those of FSH and LH, and a statistical correlation was found in two cases between FSH and prolactin. Results suggest a common releasing mechanism, which is superposed to the main mediating mechanism.

  19. Anaerobic exercise - Induced changes in serum mineral ...

    African Journals Online (AJOL)

    Anaerobic exercise, a non 02 – dependent energy metabolism leads to transient metabolic changes, which are corrected gradually by homestatic mechanism. We investigated in eight male subjects, the effects of anaerobic exercise after a day sedentary activity on serum mineral concentration. There was significant ...

  20. A ROS-dependent and Caspase-3-mediated apoptosis in sheep bronchial epithelial cells in response to Mycoplasma Ovipneumoniae infections.

    Science.gov (United States)

    Xue, Di; Li, Yanan; Jiang, Zhongjia; Deng, Guangcun; Li, Min; Liu, Xiaoming; Wang, Yujiong

    2017-05-01

    Mycoplasma Ovipneumoniae (M. ovipneumoniae) is a primary etiological agent of enzootic pneumonia in sheep and goats. It can enter and colonize ovine respiratory epithelial cells to establish an infection, which leads a serious cell death of epithelial cells. However, the nature of the interaction between pathogen of M. ovipneumoniae and host cells in the cell injury is currently not well understood. In this study, we investigated the epithelial cell apoptosis caused by an infection of M. ovipneumoniae in sheep primary air-liquid interface (ALI) epithelial cultures. The results showed that M. ovipneumoniae could specifically bind to ciliated cells at early stage of infection. Flow cytometric analysis demonstrated that an infection of M. ovipneumoniae induced a time-dependent cell apoptotic cell death, accompanied with an increased production of extracellular nitric oxide (NO), intracellular reactive oxygen species (ROS) production and activation of caspase-3 signaling in sheep bronchial epithelial cells. The induced cell apoptosis was further confirmed by a transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. Interestingly, the M. ovipneumoniae-induced apoptosis and activation of caspase-3 were correlated with the production of ROS but not NO. Mechanistically, M. ovipneumoniae-induced cell apoptosis was mediated by a mechanism by increasing the expression of phosphorylation of p38 and pro-apoptotic proteins, and activating caspase-3, caspase-8 and poly ADP-ribose polymerase (PARP) cleavage. These results suggest a ROS-dependent and caspase-3-mediated cell apoptosis in sheep bronchial epithelial cells in response to M. ovipneumoniae infections. Copyright © 2017. Published by Elsevier B.V.

  1. An N-methyl-D-aspartate receptor mediated large, low-frequency, spontaneous excitatory postsynaptic current in neonatal rat spinal dorsal horn neurons.

    Science.gov (United States)

    Thomson, L M; Zeng, J; Terman, G W

    2006-09-01

    Examples of spontaneous oscillating neural activity contributing to both pathological and physiological states are abundant throughout the CNS. Here we report a spontaneous oscillating intermittent synaptic current located in lamina I of the neonatal rat spinal cord dorsal horn. The spontaneous oscillating intermittent synaptic current is characterized by its large amplitude, slow decay time, and low-frequency. We demonstrate that post-synaptic N-methyl-D-aspartate receptors (NMDARs) mediate the spontaneous oscillating intermittent synaptic current, as it is inhibited by magnesium, bath-applied d-2-amino-5-phosphonovalerate (APV), or intracellular MK-801. The NR2B subunit of the NMDAR appears important to this phenomenon, as the NR2B subunit selective NMDAR antagonist, alpha-(4-hydroxphenyl)-beta-methyl-4-benzyl-1-piperidineethanol tartrate (ifenprodil), also partially inhibited the spontaneous oscillating intermittent synaptic current. Inhibition of spontaneous glutamate release by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5] enkephalin-ol (DAMGO) inhibited the spontaneous oscillating intermittent synaptic current frequency. Marked inhibition of spontaneous oscillating intermittent synaptic current frequency by tetrodotoxin (TTX), but not post-synaptic N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), suggests that the glutamate release important to the spontaneous oscillating intermittent synaptic current is dependent on active neural processes. Conversely, increasing dorsal horn synaptic glutamate release by GABAA or glycine inhibition increased spontaneous oscillating intermittent synaptic current frequency. Moreover, inhibiting glutamate transporters with threo-beta-benzyloxyaspartic acid (DL-TBOA) increased spontaneous oscillating intermittent synaptic current frequency and decay time. A possible functional role of this spontaneous NMDAR-mediated

  2. Circadian clocks govern calorie restriction-mediated life span extension through BMAL1- and IGF-1-dependent mechanisms.

    Science.gov (United States)

    Patel, Sonal A; Chaudhari, Amol; Gupta, Richa; Velingkaar, Nikkhil; Kondratov, Roman V

    2016-04-01

    Calorie restriction (CR) increases longevity in many species by unknown mechanisms. The circadian clock was proposed as a potential mediator of CR. Deficiency of the core component of the circadian clock-transcriptional factor BMAL1 (brain and muscle ARNT [aryl hydrocarbon receptor nuclear translocator]-like protein 1)-results in accelerated aging. Here we investigated the role of BMAL1 in mechanisms of CR. The 30% CR diet increased the life span of wild-type (WT) mice by 20% compared to mice on anad libitum(AL) diet but failed to increase life span ofBmal1(-/-)mice. BMAL1 deficiency impaired CR-mediated changes in the plasma levels of IGF-1 and insulin. We detected a statistically significantly reduction of IGF-1 in CRvs.AL by 50 to 70% in WT mice at several daily time points tested, while inBmal1(-/-)the reduction was not significant. Insulin levels in WT were reduced by 5 to 9%, whileBmal1(-/-)induced it by 10 to 35% at all time points tested. CR up-regulated the daily average expression ofBmal1(by 150%) and its downstream target genesPeriods(by 470% forPer1and by 130% forPer2). We propose that BMAL1 is an important mediator of CR, and activation of BMAL1 might link CR mechanisms with biologic clocks.-Patel, S. A., Chaudhari, A., Gupta, R., Velingkaar, N., Kondratov, R. V. Circadian clocks govern calorie restriction-mediated life span extension through BMAL1- and IGF-1-dependent mechanisms. © FASEB.

  3. High-pass filtering and dynamic gain regulation enhance vertical bursts transmission along the mossy fiber pathway of cerebellum

    Directory of Open Access Journals (Sweden)

    Jonathan Mapelli

    2010-05-01

    Full Text Available Signal elaboration in the cerebellum mossy fiber input pathway presents controversial aspects, especially concerning gain regulation and the spot-like (rather than beam-like appearance of granular-to-molecular layer transmission. By using voltage-sensitive dye (VSD imaging in rat cerebellar slices (Mapelli et al., 2010, we found that mossy fiber bursts optimally excited the granular layer above ~50 Hz and the overlaying molecular layer above ~100 Hz, thus generating a cascade of high-pass filters. NMDA receptors enhanced transmission in the granular, while GABA-A receptors depressed transmission in both the granular and molecular layer. Burst transmission gain was controlled through a dynamic frequency-dependent involvement of these receptors. Moreover, while high-frequency transmission was enhanced along vertical lines connecting the granular to molecular layer, no high-frequency enhancement was observed along the parallel fiber axis in the molecular layer. This was probably due to the stronger effect of Purkinje cell GABA-A receptor-mediated inhibition occurring along the parallel fibers than along the granule cell axon ascending branch. The consequent amplification of burst responses along vertical transmission lines could explain the spot-like activation of Purkinje cells observed following punctuate stimulation in vivo .

  4. Changes in serum biochemical factors associated with opium addiction after addiction desertion.

    Science.gov (United States)

    Afarinesh, Mohammad Reza; Haghpanah, Tahereh; Divsalar, Kouros; Dehyadegary, Elham; Shaikh-Aleslami, Azar; Mahmoodi, Majid

    2014-01-01

    The long time use of opium has some effects on serums biochemical factors, the determination of this variation is a new approach in understanding off addiction and relive of drug abuser health. Hence in this study, these indicators in person who were withdrawing of opium have been studied. In this cross-sectional study bloods biochemical factors such as fasting blood sugar (FBS), sodium (Na), calcium (Ca), uric acid (UA), blood urea nitrogen (BUN), creatinine, cholesterol, total protein, and fibrinogen in three groups serum were studied: (1) Who had been permanent opium users more than 2 years (case). (2) Dependent person who has taken one month addiction withdrawal course (control). (3) A healthy group that had been demographically similar to the other groups. According to these study findings, FBS serum level in the case group is lower than control group. Serum level of Na, creatinine, and blood triglyceride (TG) in case study are higher than group control. Concentration of potassium, Ca, UA, BUN, cholesterol, total serum protein, fibrinogen, and thrombin time in case study and group control showed no significant difference. Also, in withdrawing case serum level of Na, Ca, UA, BUN, creatinine, and TG significantly increase and thrombin time decrease. According to this study not only the longtime use of opium but also opium with drawerin opium dependent people can change their serum biochemical factors. So recognition, treatment, and prevention of this change could be a new step in improving of health and condition of patients.

  5. Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells.

    Science.gov (United States)

    Yang, Eun Sun; Yang, Joon-Hyuck; Park, Ji Eun; Park, Jeen-Woo

    2005-01-01

    Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP+ -dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.

  6. Distinct kinetics of inhibitory currents in thalamocortical neurons that arise from dendritic or axonal origin.

    Directory of Open Access Journals (Sweden)

    Sunggu Yang

    Full Text Available Thalamocortical neurons in the dorsal lateral geniculate nucleus (dLGN transfer visual information from retina to primary visual cortex. This information is modulated by inhibitory input arising from local interneurons and thalamic reticular nucleus (TRN neurons, leading to alterations of receptive field properties of thalamocortical neurons. Local GABAergic interneurons provide two distinct synaptic outputs: axonal (F1 terminals and dendritic (F2 terminals onto dLGN thalamocortical neurons. By contrast, TRN neurons provide only axonal output (F1 terminals onto dLGN thalamocortical neurons. It is unclear if GABAA receptor-mediated currents originating from F1 and F2 terminals have different characteristics. In the present study, we examined multiple characteristics (rise time, slope, halfwidth and decay τ of GABAA receptor-mediated miniature inhibitory postsynaptic synaptic currents (mIPSCs originating from F1 and F2 terminals. The mIPSCs arising from F2 terminals showed slower kinetics relative to those from F1 terminals. Such differential kinetics of GABAAR-mediated responses could be an important role in temporal coding of visual signals.

  7. Dioxin-like activities in serum across European and Inuit populations

    Science.gov (United States)

    Long, Manhai; Andersen, Birgitte S; Lindh, Christian H; Hagmar, Lars; Giwercman, Aleksander; Manicardi, Gian-Carlo; Bizzaro, Davide; Spanò, Marcello; Toft, Gunnar; Pedersen, Henning S; Zvyezday, Valentyna; Bonde, Jens Peter; Bonefeld-Jorgensen, Eva C

    2006-01-01

    Background Persistent organic pollutants (POPs) such as polychlorinated dibenzo-p-dioxins/furans, polychlorinated biphenyls (PCBs) and organochlorine pesticides can cause a series of adverse effects on e.g. reproduction in animals and humans, many of which involve the aryl hydrocarbon receptor (AhR). The aim of the present study was to compare the integrated serum level of AhR mediated activity among European and Inuit populations, and evaluate whether the activity was associated to the selected POP markers, 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE). Methods The study included 338 males from Greenland (Inuit's), Sweden, Warsaw (Poland) and Kharkiv (Ukraine). The AhR transactivity of serum extracts alone (AhRag) and competitive AhR activity (AhRcomp) upon co-exposure with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were determined in the lipophilic serum fraction containing the POPs using the AhR mediated luciferase reporter Hepa1.12cR cell assay. Results The European groups showed higher median level of AhR-TEQ (TCDD toxic equivalents) compared to the Inuit's, whereas higher incidence of Inuits sample further induced AhRcomp activity. Neither AhRag nor AhR-TEQ were correlated to CB-153 or p,p'-DDE for any of the study groups. Multiple regressions showed a significant heterogeneity of association between the CB-153 and the AhRcomp across the study groups, and accordingly a negative association between AhRcomp and CB-153 was found for the Kharkiv group. Conclusion No consistent correlation between AhR activities and two POP markers was found. Although the difference of AhRag between European and Inuit men could not be explained by CB-153 or p,p'-DDE levels alone, we believe that the variation of AhR serum activity reflects different pattern of POP exposure, genetics and/or life style factors. PMID:16725033

  8. GABA-mediated synchronization in the human neocortex: elevations in extracellular potassium and presynaptic mechanisms.

    Science.gov (United States)

    Louvel, J; Papatheodoropoulos, C; Siniscalchi, A; Kurcewicz, I; Pumain, R; Devaux, B; Turak, B; Esposito, V; Villemeure, J G; Avoli, M

    2001-01-01

    Field potential and extracellular [K(+)] ([K(+)](o)) recordings were made in the human neocortex in an in vitro slice preparation to study the synchronous activity that occurs in the presence of 4-aminopyridine (50 microM) and ionotropic excitatory amino acid receptor antagonists. Under these experimental conditions, negative or negative-positive field potentials accompanied by rises in [K(+)](o) (up to 4.1 mM from a baseline of 3.25 mM) occurred spontaneously at intervals of 3-27 s. Both field potentials and [K(+)](o) elevations were largest at approximately 1000 microm from the pia. Similar events were induced by neocortical electrical stimuli. Application of medium containing low [Ca(2+)]/high [Mg(2+)] (n=3 slices), antagonism of the GABA(A) receptor (n=7) or mu-opioid receptor activation (n=4) abolished these events. Hence, they represented network, GABA-mediated potentials mainly reflecting the activation of type A receptors following GABA release from interneurons. The GABA(B) receptor agonist baclofen (10-100 microM, n=11) reduced and abolished the GABA-mediated potentials (ID(50)=18 microM). Baclofen effects were antagonized by the GABA(B) receptor antagonist CGP 35348 (0.1-1 mM, n=6; ID(50)=0.19 mM). CGP 38345 application to control medium increased the amplitude of the GABA-mediated potentials and the concomitant [K(+)](o) rises without modifying their rate of occurrence. The GABA-mediated potentials were not influenced by the broad-spectrum metabotropic glutamate agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (100 microM, n=10), but decreased in rate with the group I receptor agonist (S)-3,5-dihydroxyphenylglycine (10-100 microM, n=9). Our data indicate that human neocortical networks challenged with 4-aminopyridine generate glutamatergic-independent, GABA-mediated potentials that are modulated by mu-opioid and GABA(B) receptors presumably located on interneuron terminals. These events are associated with [K(+)](o) elevations that may

  9. A single bout of high-intensity interval training improves motor skill retention in individuals with stroke

    DEFF Research Database (Denmark)

    Nepveu, Jean-Francois; Thiel, Alexander; Tang, Ada

    2017-01-01

    imbalances in GABAA-mediated short-interval intracortical inhibition but changes in other markers of excitability were not statistically significant. The group that performed high-intensity interval training showed a better retention of the motor skill. CONCLUSIONS: The performance of a maximal graded...

  10. Does mortality risk of cigarette smoking depend on serum concentrations of persistent organic pollutants? Prospective investigation of the vasculature in Uppsala seniors (PIVUS study.

    Directory of Open Access Journals (Sweden)

    Duk-Hee Lee

    Full Text Available Cigarette smoking is an important cause of preventable death globally, but associations between smoking and mortality vary substantially across country and calendar time. Although methodological biases have been discussed, it is biologically plausible that persistent organic pollutants (POPs like polychlorinated biphenyls (PCBs and organochlorine (OC pesticides can affect this association. This study was performed to evaluate if associations of cigarette smoking with mortality were modified by serum concentrations of PCBs and OC pesticides. We evaluated cigarette smoking in 111 total deaths among 986 men and women aged 70 years in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS with mean follow-up for 7.7 years. The association between cigarette smoking and total mortality depended on serum concentration of PCBs and OC pesticides (P value for interaction = 0.02. Among participants in the highest tertile of the serum POPs summary score, former and current smokers had 3.7 (95% CI, 1.5-9.3 and 6.4 (95% CI, 2.3-17.7 times higher mortality hazard, respectively, than never smokers. In contrast, the association between cigarette smoking and total mortality among participants in the lowest tertile of the serum POPs summary score was much weaker and statistically non-significant. The strong smoking-mortality association observed among elderly people with high POPs was mainly driven by low risk of mortality among never smokers with high POPs. As smoking is increasing in many low-income and middle-income countries and POPs contamination is a continuing problem in these areas, the interactions between these two important health-related issues should be considered in future research.

  11. Serum under-carboxylated osteocalcin levels in women with polycystic ovary syndrome: weight-dependent relationships with endocrine and metabolic traits

    Directory of Open Access Journals (Sweden)

    Pepene Carmen E

    2013-01-01

    Full Text Available Abstract Background Under-carboxylated osteocalcin (ucOC, the precursor substrate of bone biomarker OC is a potent regulator of energy metabolism by promoting insulin production and adiponectin synthesis and decreasing fat stores. The aim of the present study was to point out the potential role of ucOC in the physiopathology of polycystic ovary syndrome (PCOS, a common disorder defined by the constellation of anovulation, insulinresistance, hyperinsulinemia, obesity and androgen excess. Methods In this prospective case–control investigation, 78 young premenopausal women, i.e. 52 PCOS patients and 26 age- and body mass index (BMI-matched healthy controls, were successively enrolled. Recruitment of PCOS patients was performed according to Androgen Excess-Polycystic Ovary Syndrome (AE-PCOS Society 2006 criteria. All study participants were subjected to clinical examination, whole-body composition assessment and measurements of serum ucOC, OC (1-49, glucose and lipids, insulin, total testosterone (TT, estradiol, sex-hormone binding globulin (SHBG, high-sensitivity C-reactive protein (Hs-CRP and β-CrossLaps. Results BMI-stratified multivariate analysis revealed significantly higher ucOC levels in PCOS vs. controls in lean (p = 0.001 but not overweight and obese study participants (p = 0.456. Notably, a positive correlation between ucOC and TT (p = 0.018, calculated free testosterone (cFT, p = 0.028 and serum insulin (p = 0.036, respectively, was found to be confined to the lean analysis subgroup. Furthermore, in stepwise multiple regression models, β-CrossLaps and cFT were able to predict 46.71% of serum ucOC variability. (1-43/49OC failed to be significantly associated to any PCOS trait. Conclusions Circulating ucOC concentration is related to key endocrine PCOS characteristics in a weight-dependent manner. Within the bone-pancreas loop, high ucOC may favor insulin release in lean hyperandrogenic women to compensate for

  12. The levels of triglyceride and total cholesterol in methamphetamine dependence.

    Science.gov (United States)

    Zhang, Meijuan; Lv, Dezhao; Zhou, Wu; Ji, Lili; Zhou, Beibei; Chen, Han; Gu, Yingying; Zhao, Jiyun; He, Jincai

    2017-04-01

    The serum triglyceride (TG) and total cholesterol (TC) levels have been reported altered in the traditional drug-dependence (such as marijuana and heroin). However, studies assessing the relationships among serum TC, TG, and methamphetamine (MA)-dependence have not been described well. In this study, our aim is to explore the serum TG and TC levels in large sample of MA-dependent patients. A retrospective study was conducted in 938 MA-dependent patients who were recruited between February 2, 2008 and March 11, 2013, with social characteristics and drug-dependence history (duration of MA use, routes of drug administration, and daily dose were collected). Then, the serum levels of TC, TG, glucose (GLU), body mass index (BMI), and blood pressure were measured among the participants. Meanwhile, 985 age- and gender-matched healthy people in the physical examination center were selected as control group. Compared with the control group, significant decreases of TC, TG, GLU, and BMI were observed in MA-dependent patients (P < 0.05). Besides, we found that the daily dose of MA use was associated with TC (β = -0.079, P = 0.015) and the duration of MA use was independently related to BMI (β = -0.071, P = 0.031). This study demonstrated that the levels of TC, TG, GLU, and BMI factors altered in the MA-dependent patients. In addition, there is a negative association between MA dependence and TC and BMI.

  13. Curcumin attenuates oxidative stress induced NFκB mediated inflammation and endoplasmic reticulum dependent apoptosis of splenocytes in diabetes.

    Science.gov (United States)

    Rashid, Kahkashan; Chowdhury, Sayantani; Ghosh, Sumit; Sil, Parames C

    2017-11-01

    The present study was aimed to determine the curative role of curcumin against diabetes induced oxidative stress and its associated splenic complications. Diabetes was induced in the experimental rats via the intraperitoneal administration of a single dose of STZ (65mgkg -1 body weight). Increased blood glucose and intracellular ROS levels along with decreased body weight, the activity of cellular antioxidant enzymes and GSH/GSSG ratio were observed in the diabetic animals. Histological assessment showed white pulp depletion and damaged spleen anatomy in these animals. Oral administration of curcumin at a dose of 100mgkg -1 body weight daily for 8weeks, however, restored these alterations. Investigation of the mechanism of hyperglycemia induced oxidative stress mediated inflammation showed upregulation of inflammatory cytokines, chemokines, adhesion molecules and increased translocation of NFκB into the nucleus. Moreover, ER stress dependent cell death showed induction of eIF2α and CHOP mediated signalling pathways as well as increment in the expression of GRP78, Caspase-12, Calpain-1, phospho JNK, phospho p38 and phospho p53 in the diabetic group. Alteration of Bax/Bcl-2 ratio; disruption of mitochondrial membrane potential, release of cytochrome-C from mitochondria and upregulation of caspase 3 along with the formation of characteristic DNA ladder in the diabetic animals suggest the involvement of mitochondria dependent apoptotic pathway in the splenic cells. Treatment with curcumin could, however, protect cells from inflammatory damage and ER as well as mitochondrial apoptotic death by restoring the alterations of these parameters. Our results suggest that curcumin has the potential to act as an anti-diabetic, anti-oxidant, anti-inflammatory and anti-apoptotic therapeutic against diabetes mediated splenic damage. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages.

    Science.gov (United States)

    Datta, Debika; Khatri, Preeti; Banerjee, Chaitali; Singh, Ambika; Meena, Ramavatar; Saha, Dhira Rani; Raman, Rajagopal; Rajamani, Paulraj; Mitra, Abhijit; Mazumder, Shibnath

    2016-01-01

    Mycobacterium fortuitum causes 'mycobacteriosis' in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.

  15. Inhibition of fibroblast growth by Notch1 signaling is mediated by induction of Wnt11-dependent WISP-1.

    Directory of Open Access Journals (Sweden)

    Zhao-Jun Liu

    Full Text Available Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM. They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox embryonic fibroblasts (MEFs. Notch1-deficient (Notch1(-/- MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1 in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441, which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1. Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4 in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

  16. Spermidine mediates degradation of ornithine decarboxylase by a non-lysosomal, ubiquitin-independent mechanism

    International Nuclear Information System (INIS)

    Glass, J.R.; Gerner, E.W.

    1987-01-01

    The mechanism of spermidine-induced ornithine decarboxylase (OCD, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum-free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine. Treatment of cells with 10 μM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [ 35 S]methionine-labeled ODC and separation by two-dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine-mediated inactivation of ODC occurred in a temperature-dependent manner exhibiting pseudo-first-order kinetics over a temperature range of 22-37 0 C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22 0 C for 3 hours with 10 μ M spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents had no effect of ODC degradation. ODC turnover was not dependent on ubiquitin-dependent proteolysis. These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non-lysosomal, ubiquitin-independent pathway

  17. Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab.

    Science.gov (United States)

    Fujii, Rika; Friedman, Eitan R; Richards, Jacob; Tsang, Kwong Y; Heery, Christopher R; Schlom, Jeffrey; Hodge, James W

    2016-06-07

    Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC.

  18. Shape-dependent bactericidal activity of copper oxide nanoparticle mediated by DNA and membrane damage

    International Nuclear Information System (INIS)

    Laha, Dipranjan; Pramanik, Arindam; Laskar, Aparna; Jana, Madhurya; Pramanik, Panchanan; Karmakar, Parimal

    2014-01-01

    Highlights: • Spherical and sheet shaped copper oxide nanoparticles were synthesized. • Physical characterizations of these nanoparticles were done by TEM, DLS, XRD, FTIR. • They showed shape dependent antibacterial activity on different bacterial strain. • They induced both membrane damage and ROS mediated DNA damage in bacteria. - Abstract: In this work, we synthesized spherical and sheet shaped copper oxide nanoparticles and their physical characterizations were done by the X-ray diffraction, fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering. The antibacterial activity of these nanoparticles was determined on both gram positive and gram negative bacterial. Spherical shaped copper oxide nanoparticles showed more antibacterial property on gram positive bacteria where as sheet shaped copper oxide nanoparticles are more active on gram negative bacteria. We also demonstrated that copper oxide nanoparticles produced reactive oxygen species in both gram negative and gram positive bacteria. Furthermore, they induced membrane damage as determined by atomic force microscopy and scanning electron microscopy. Thus production of and membrane damage are major mechanisms of the bactericidal activity of these copper oxide nanoparticles. Finally it was concluded that antibacterial activity of nanoparticles depend on physicochemical properties of copper oxide nanoparticles and bacterial strain

  19. Shape-dependent bactericidal activity of copper oxide nanoparticle mediated by DNA and membrane damage

    Energy Technology Data Exchange (ETDEWEB)

    Laha, Dipranjan; Pramanik, Arindam [Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032 (India); Laskar, Aparna [CSIR-Indian Institute of Chemical Biology, Kolkata 700032 (India); Jana, Madhurya [Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032 (India); Pramanik, Panchanan [Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India); Karmakar, Parimal, E-mail: pkarmakar_28@yahoo.co.in [Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032 (India)

    2014-11-15

    Highlights: • Spherical and sheet shaped copper oxide nanoparticles were synthesized. • Physical characterizations of these nanoparticles were done by TEM, DLS, XRD, FTIR. • They showed shape dependent antibacterial activity on different bacterial strain. • They induced both membrane damage and ROS mediated DNA damage in bacteria. - Abstract: In this work, we synthesized spherical and sheet shaped copper oxide nanoparticles and their physical characterizations were done by the X-ray diffraction, fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering. The antibacterial activity of these nanoparticles was determined on both gram positive and gram negative bacterial. Spherical shaped copper oxide nanoparticles showed more antibacterial property on gram positive bacteria where as sheet shaped copper oxide nanoparticles are more active on gram negative bacteria. We also demonstrated that copper oxide nanoparticles produced reactive oxygen species in both gram negative and gram positive bacteria. Furthermore, they induced membrane damage as determined by atomic force microscopy and scanning electron microscopy. Thus production of and membrane damage are major mechanisms of the bactericidal activity of these copper oxide nanoparticles. Finally it was concluded that antibacterial activity of nanoparticles depend on physicochemical properties of copper oxide nanoparticles and bacterial strain.

  20. Serum creatine kinase and lactate dehydrogenase activities in ...

    African Journals Online (AJOL)

    ... in thyroid function are common endocrine disorders affecting 5-10% of individuals over ... Key words: Hyperthyroidism, hypothyroidism, lactate dehydrogenase, serum creatine kinase ... individuals depends on age, race, lean body mass and physical activity. ... measured by radioimmunoassay on AXSYM System (Abbott.