Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

Efeito da hiperalimentação na lactação sobre a regulação da adiposidade corporal e sinalização da grelina no tecido adiposo branco de camundongos jovens e adultos

A obesidade é um dos maiores problemas de saúde pública que cresce em todo o mundo, resultante de um desequilíbrio entre ingestão alimentar e gasto energético. O aumento da adiposidade leva ao desenvolvimento de alterações funcionais. Pode-se dizer que a obesidade é o principal fator de risco para o desenvolvimento de doenças crônicas de maior prevalência como dislipidemias, doenças cardiovasculares e diabetes do tipo 2, acarretando na redução da qualidade e expectativa de vida. A Grelina é um hormônio sintetizado pelo estômago, que atua em diferentes tecidos através de um receptor específico (GHS-R1a), incluindo hipotálamo e tecido adiposo. A grelina tem uma ação direta sobre a regulação hipotalâmica da ingestão alimentar, induzindo um efeito orexígeno. Por outro lado, a grelina também modula o armazenamento de energia nos adipócitos. Esta dupla ação sugere que este hormônio pode atuar como uma ligação entre o sistema nervoso central e mecanismos periféricos. Portanto, considerando que a hiperalimentação neonatal induz obesidade na idade adulta por mecanismos desconhecidos, neste estudo foram pesquisados os efeitos da hiperalimentação no início da vida sobre o desenvolvimento da obesidade e, em particular, a sinalização da grelina no tecido adiposo em ratos jovens e adultos. Foram utilizados camundongos Swiss hiperalimentados através do modelo de redução da ninhada. Para induzir a hiperalimentação as ninhadas foram reduzidas a 3 filhotes machos por lactante no 30 dia de vida pós-natal. As ninhadas controles foram ajustadas em 9 filhotes por lactante. Foram avaliados parâmetros antropométricos como: massa corporal e massa do tecido adiposo visceral. A glicemia de jejum foi avaliada utilizando glicosímetro e fitas teste. A análise do conteúdo das proteínas envolvidas na via de sinalização da grelina foram detectadas pelo método de Western Blotting. Os grupos controle (C) e hiperalimentado (H) foram estudados aos 21 e 180 dias de vida. Os dados demonstram que a hipernutrição no início da vida induz um aumento significativo no peso corporal dos camundongos jovens, começando aos 10 dias, e este aumento de peso persistiu até à idade adulta (180 dias de idade). A glicemia e o peso da gordura visceral foram significativamente maiores no grupo hiperalimentado aos 21 e 180 dias, quando comparado com o grupo controle. Os níveis plasmáticos de grelina acilada apresentaram uma redução de 70% nos animais jovens e 49% adultos obesos. Além disso, no tecido adiposo branco, observamos um maior conteúdo (242%) do receptor de grelina (GHSR1a) nos animais hiperalimentados com 21 dias, e este aumento foi associado à modulação positiva do conteúdo e fosforilação de proteínas envolvidas no estoque e utilização de energia celular, tais como AKT, PI3K, AMPK, GLUT-4, e CPT1. No entanto, ao chegar à idade adulta os animais hiperalimentados não apresentaram diferença significativa no conteúdo de GHS-R1a e das proteínas AKT, PI3K, AMPK, GLUT-4, e CPT1. O conteúdo de PPARɣ foi menor no grupo obeso aos 21e 180 dias. Basicamente, mostramos que o metabolismo do tecido adiposo está alterado na obesidade adquirida no início da vida e, provavelmente, devido a essa modificação, ocorre um novo padrão da via de sinalização da grelina.

Adaptive control of event integration: evidence from event-related potentials

We investigated whether it is possible to control the temporal window of attention used to rapidly integrate visual information. To study the underlying neural mechanisms, we recorded ERPs in an attentional blink task, known to elicit Lag-1 sparing. Lag-1 sparing fosters joint integration of the two targets, evidenced by increased order errors. Short versus long integration windows were induced by showing participants mostly fast or slow stimuli. Participants expecting slow speed used a longer integration window, increasing joint integration. Difference waves showed an early (200 ms post-T2) negative and a late positive modulation (390 ms) in the fast group, but not in the slow group. The modulations suggest the creation of a separate event for T2, which is not needed in the slow group, where targets were often jointly integrated. This suggests that attention can be guided by global expectations of presentation speed within tens of milliseconds.

Metabolism of anthocyanins by human gut microflora and their influence on gut bacterial growth

Consumption of anthocyanins has been related with beneficial health effects. However, bioavailability studies have shown low concentration of anthocyanins in plasma and urine. In this study, we have investigated the bacterial-dependent metabolism of malvidin-3-glucoside, gallic acid and a mixture of anthocyanins using a pH-controlled, stirred, batch-culture fermentation system reflective of the distal human large intestine conditions. Most anthocyanins have disappeared after 5 h incubation while gallic acid remained constant through the first 5 h and was almost completely degraded following 24 h of fermentation. Incubation of malvidin-3-glucoside with fecal bacteria mainly resulted in the formation of syringic acid, while the mixture of anthocyanins resulted in formation of gallic, syringic and p-coumaric acids. All the anthocyanins tested enhanced significantly the growth of Bif idobacterium spp. and Lactobacillus−Enterococcus spp. These results suggest that anthocyanins and their metabolites may exert a positive modulation of the intestinal bacterial population.

Actions of octocoral and tobacco cembranoids on nicotinic receptors

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies. (C) 2009 Elsevier Ltd. All rights reserved.

Mode of cembranoid action on embryonic muscle acetylcholine receptor

The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 mu M EUAC with 50 mu M, 100 N, or 500 mu M carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 mu M CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 N) obtained with 100 mu M and 500 mu M CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations. (c) 2007 Wiley-Liss, Inc.

A soy-based product fermented by Enterococcus faecium and Lactobacillus helveticus inhibits the development of murine breast adenocarcinoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The involvement of anti-inflammatory protein, Annexin A1, in ocular toxoplasmosis

Purpose: The aim of this study was to evaluate the expression of the protein annexin A1 (ANXA1), a potent endogenous regulator of the inflammatory process, in ocular toxoplasmosis. Methods: C57BL/6 female mice were infected using intravitreal injections of either 10 6 tachyzoites of Toxoplasma gondii (RH strain; T. gondii) or PBS only (control groups). After 24, 48, and 72 h, animals were sacrificed and their eyes were harvested for histopathological, immunohistochemical, and ultrastructural immunocytochemical analysis of ANXA1. Human retinal pigment epithelial (RPE) cells (ARPE-19) were infected in vitro with T. gondii and collected after 60, 120, 240 min, and 24 h. Results: Compared with non-infected eyes, an intense inflammatory response was observed in the anterior (24 h after infection) and posterior segments (72 h after infection) of the infected eye, characterized by neutrophil infiltration and by the presence of tachyzoites and their consequent destruction along with disorganization of normal retina architecture and RPE vacuolization. T. gondii infection was associated with a significant increase of ANXA1 expression in the neutrophils at 24, 48, and 72 h, and in the RPE at 48 and 72 h. In vitro studies confirmed an upregulation of ANXA1 levels in RPE cells, after 60 and 120 min of infection with T. gondii. Conclusions: The positive modulation of endogenous ANXA1 in the inflammatory and RPE cells during T. gondii infection suggests that this protein may serve as a therapeutic target in ocular toxoplasmosis. © 2012 Molecular Vision.

Seeing with the eyes shut: Neural basis of enhanced imagery following ayahuasca ingestion

The hallucinogenic brew Ayahuasca, a rich source of serotonergic agonists and reuptake inhibitors, has been used for ages by Amazonian populations during religious ceremonies. Among all perceptual changes induced by Ayahuasca, the most remarkable are vivid seeings. During such seeings, users report potent imagery. Using functional magnetic resonance imaging during a closed-eyes imagery task, we found that Ayahuasca produces a robust increase in the activation of several occipital, temporal, and frontal areas. In the primary visual area, the effect was comparable in magnitude to the activation levels of natural image with the eyes open. Importantly, this effect was specifically correlated with the occurrence of individual perceptual changes measured by psychiatric scales. The activity of cortical areas BA30 and BA37, known to be involved with episodic memory and the processing of contextual associations, was also potentiated by Ayahuasca intake during imagery. Finally, we detected a positive modulation by Ayahuasca of BA 10, a frontal area involved with intentional prospective imagination, working memory and the processing of information from internal sources. Therefore, our results indicate that Ayahuasca seeings stem from the activation of an extensive network generally involved with vision, memory, and intention. By boosting the intensity of recalled images to the same level of natural image, Ayahuasca lends a status of reality to inner experiences. It is therefore understandable why Ayahuasca was culturally selected over many centuries by rain forest shamans to facilitate mystical revelations of visual nature. Hum Brain Mapp, 2012. (c) 2011 Wiley Periodicals, Inc.

Affect, attention, or anticipatory arousal? Human blink startle modulation in forward and backward affective conditioning

Affect modulates the blink startle reflex in the picture-viewing paradigm, however, the process responsible for reflex modulation during conditional stimuli (CSs) that have acquired valence through affective conditioning remains unclear. In Experiment 1, neutral shapes (CSs) and valenced or neutral pictures (USs) were paired in a forward (CS → US) manner. Pleasantness ratings supported affective learning of positive and negative valence. Post-acquisition, blink reflexes were larger during the pleasant and unpleasant CSs than during the neutral CS. Rather than affect, attention or anticipatory arousal were suggested as sources of startle modulation. Experiment 2 confirmed that affective learning in the picture–picture paradigm was not affected by whether the CS preceded the US. Pleasantness ratings and affective priming revealed similar extents of affective learning following forward, backward or simultaneous pairings of CSs and USs. Experiment 3 utilized a backward conditioning procedure (US → CS) to minimize effects of US anticipation. Again, blink reflexes were larger during CSs paired with valenced USs regardless of US valence implicating attention rather than anticipatory arousal or affect as the process modulating startle in this paradigm.

Electroluminescence from modulation-doped pseudomorphic AlGaAs/InGaAs/GaAs quantum wells

Low-temperature electroluminescence (EL) is observed in n-type modulation-doped AlGaAs/InGaAs/GaAs quantum well samples by applying a positive voltage between the semitransparent Au gate and alloyed Au–Ge Ohmic contacts made on the top surface of the samples. We attribute impact ionization in the InGaAs QW to the observed EL from the samples. A redshift in the EL spectra is observed with increasing gate bias. The observed redshift in the EL spectra is attributed to the band gap renormalization due to many-body effects and quantum-confined Stark effect.

The mechanisms of human renal epithelial cell modulation of autologous dendritic cell phenotype and function

Proximal tubule epithelial cells (PTEC) of the kidney line the proximal tubule downstream of the glomerulus and play a major role in the re-absorption of small molecular weight proteins that may pass through the glomerular filtration process. In the perturbed disease state PTEC also contribute to the inflammatory disease process via both positive and negative mechanisms via the production of inflammatory cytokines which chemo-attract leukocytes and the subsequent down-modulation of these cells to prevent uncontrolled inflammatory responses. It is well established that dendritic cells are responsible for the initiation and direction of adaptive immune responses. Both resident and infiltrating dendritic cells are localised within the tubulointerstitium of the renal cortex, in close apposition to PTEC, in inflammatory disease states. We previously demonstrated that inflammatory PTEC are able to modulate autologous human dendritic cell phenotype and functional responses. Here we extend these findings to characterise the mechanisms of this PTEC immune-modulation using primary human PTEC and autologous monocyte-derived dendritic cells (MoDC) as the model system. We demonstrate that PTEC express three inhibitory molecules: (i) cell surface PD-L1 that induces MoDC expression of PD-L1; (ii) intracellular IDO that maintains the expression of MoDC CD14, drives the expression of CD80, PD-L1 and IL-10 by MoDC and inhibits T cell stimulatory capacity; and (iii) soluble HLA-G (sHLA-G) that inhibits HLA-DR and induces IL-10 expression by MoDC. Collectively the results demonstrate that primary human PTEC are able to modulate autologous DC phenotype and function via multiple complex pathways. Further dissection of these pathways is essential to target therapeutic strategies in the treatment of inflammatory kidney disorders.

A new method for modulating the activity of parvalbumin-positive neurons and cerebellar Purkinje cells in vivo

Neurons can be divided into various classes according to their location, morphology, neurochemical identity and electrical properties. They form complex interconnected networks with precise roles for each cell type. GABAergic neurons expressing the calcium-binding protein parvalbumin (Pv) are mainly interneurons, which serve a coordinating function. Pv-cells modulate the activity of principal cells with high temporal precision. Abnormalities of Pv-interneuron activity in cortical areas have been linked to neuropsychiatric illnesses such as schizophrenia. Cerebellar Purkinje cells are known to be central to motor learning. They are the sole output from the layered cerebellar cortex to deep cerebellar nuclei. There are still many open questions about the precise role of Pv-neurons and Purkinje cells, many of which could be answered if one could achieve rapid, reversible cell-type specific modulation of the activity of these neurons and observe the subsequent changes at the whole-animal level. The aim of these studies was to develop a novel method for the modulation of Pv-neurons and Purkinje cells in vivo and to use this method to investigate the significance of inhibition in these neuronal types with a variety of behavioral experiments in addition to tissue autoradiography, electrophysiology and immunohistochemistry. The GABA(A) receptor γ2 subunit was ablated from Pv-neurons and Purkinje cells in four separate mouse lines. Pv-Δγ2 mice had wide-ranging behavioral alterations and increased GABA-insensitive binding indicative of an altered GABA(A) receptor composition, particularly in midbrain areas. PC-Δγ2 mice experienced little or no motor impairment despite the lack of inhibition in Purkinje cells. In Pv-Δγ2-partial rescue mice, a reversal of motor and cognitive deficits was observed in addition to restoration of the wild-type γ2F77 subunit to the reticular nucleus of thalamus and the cerebellar molecular layer. In PC-Δγ2-swap mice, zolpidem sensitivity was restored to Purkinje cells and the administration of systemic zolpidem evoked a transient motor impairment. On the basis of these results, it is concluded that this new method of cell-type specific modulation is a feasible way to modulate the activity of selected neuronal types. The importance of Purkinje cells to motor control supports previous studies, and the crucial involvement of Pv-neurons in a range of behavioral modalities is confirmed.

Hormonal modulation of secretion of immunoreactive riboflavin carrier protein by adult rat Leydig cells in vitro

Adult rat Leydig cells in culture synthesize and secrete riboflavin carrier protein (RCP) as demonstrated by [S-35]-methionine incorporation into newly synthesized proteins followed by immunoprecipitation as well as specific radioimmunoassay. LH stimulates the secretion of RCP 4-fold which could be inhibited upto 75% by an aromatase inhibitor. 8-bromo-cyclic AMP and cholera toxin could mimic the LH stimulated secretion of the carrier protein. The extent of stimulation of RCP secretion brought about by exogenous estradiol-17 beta is comparable to that of LH. The antiestrogen tamoxifen, when added along with either LH or estrogen, inhibited the stimulated levels significantly. These results show that the estrogen-inducible riboflavin carrier is secreted by Leydig cells under positive regulation of LH.

Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro

In designing and developing various biomaterials, the influence of substrate properties, like surface topography, stiffness and wettability on the cell functionality has been investigated widely. However, such study to probe into the influence of the substrate conductivity on cell fate processes is rather limited. In order to address this issue, spark plasma sintered HA-CaTiO3 (Hydroxyapatite-Calcium titanate) has been used as a model material system to showcase the effect of varying conductivity on cell functionality. Being electroactive in nature, mouse myoblast cells (C2C12) were selected as a model cell line in this study. It was inferred that myoblast adhesion/growth systematically increases with substrate conductivity due to CaTiO3 addition to HA. Importantly, parallel arrangement of myoblast cells on higher CaTiO3 containing substrates indicate that self-adjustable cell patterning can be achieved on conductive biomaterials. Furthermore, enhanced myoblast assembly and myotube formation were recorded after 5 days of serum starvation. Overall, the present study conclusively establishes the positive impact of the substrate conductivity towards cell proliferation and differentiation as well as confirms the efficacy of HA-CaTiO3 biocomposites as conductive platforms to facilitate the growth, orientation and fusion of myoblasts, even when cultured in the absence of external electric field.