RESUMEN
Hydrogels are supramolecular assemblies with both solute transport properties like liquids and mechanical properties like elastomers. To date, every type of biomolecules except ribonucleic acid (RNA), is capable of forming a hydrogel. Here, we report an RNA that forms a hydrogel by self-assembly. This RNA is originally identified by systematic evolution of ligands by exponential enrichment (SELEX) to enhance the activity of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors as a potential RNA drug for the treatment of cognitive disorders. The RNA hydrogel exhibits an elastic modulus plateau on the order of 102 Pa and shows dynamic RNA chain interactions with relaxation behaviors similar to living wormlike micellar solutions. Small-angle X-ray scattering and cryogenic electron microscopy characterization support the RNA network structures. By sequence mutation and rheological measurements, we reveal two key sequence motifs in the RNA responsible for intermolecular recognition and the formation of a polymer network by self-assembly.
Asunto(s)
Aptámeros de Nucleótidos , Hidrogeles , Ensayo de Materiales , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico , Aptámeros de Nucleótidos/síntesis química , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Células HEK293 , Humanos , Hidrogeles/síntesis química , Hidrogeles/química , Hidrogeles/farmacología , Técnica SELEX de Producción de Aptámeros/métodos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728-4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.
Asunto(s)
Mutación Puntual , Receptores AMPA/química , Receptores AMPA/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Sitio Alostérico , Animales , Sitios de Unión , Células HEK293 , Humanos , Cinética , Ligandos , Modelos Teóricos , Técnicas de Placa-Clamp , Unión Proteica , Estructura Terciaria de Proteína , RatasRESUMEN
Glutamate receptors mediate the majority of excitatory synaptic transmission in the central nervous system, and excessive stimulation of these receptors is involved in a variety of neurological disorders and neuronal damage from stroke. The development of new subtype-specific antagonists would be of considerable therapeutic interest. Natural products can provide important new lead compounds for drug discovery. The only natural product known to inhibit glutamate receptors competitively is (-)-kaitocephalin, which was isolated from the fungus Eupenicillium shearii and found to protect CNS neurons from excitotoxicity. Previous work has shown that it is a potent antagonist of some subtypes of glutamate receptors (AMPA and NMDA, but not kainate). The structure of kaitocephalin bound to the ligand binding domain of the AMPA receptor subtype, GluA2, is reported here. The structure suggests how kaitocephalin can be used as a scaffold to develop more selective and high affinity antagonists for glutamate receptors.
Asunto(s)
Pirroles/química , Receptores AMPA/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Animales , Sitios de Unión , Encéfalo/metabolismo , Cristalografía por Rayos X , Ácido Glutámico/química , Concentración 50 Inhibidora , Ligandos , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , RatasRESUMEN
In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.
Asunto(s)
Calcio/química , Neuronas/patología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Animales , Apoptosis , Señalización del Calcio , Muerte Celular , Embrión de Pollo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Fosforilación , Ratas , Ratas Long-Evans , Ratas Wistar , Receptores de Glutamato/metabolismo , Retina/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
The conformational changes in the agonist binding domain of the glycine-binding GluN1 and glutamate-binding GluN2A subunits of the N-methyl D-aspartic acid receptor upon binding agonists of varying efficacy have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists; however, no significant changes in the cleft closure are observed between partial and full agonists. This is consistent with the previously reported crystal structures for the isolated agonist binding domain of this receptor. Additionally, the LRET-based distances show that the agonist binding domain of the glutamate-binding GluN2A subunit exhibits a graded cleft closure with the extent of cleft closure being proportional to the extent of activation, indicating that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.
Asunto(s)
Receptores de N-Metil-D-Aspartato/química , Animales , Cristalografía por Rayos X/métodos , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/farmacología , Enlace de Hidrógeno , Modelos Moleculares , Neurotransmisores/química , Técnicas de Placa-Clamp , Unión Proteica , Conformación Proteica , Receptores de Ácido Kaínico/química , Xenopus laevis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/químicaRESUMEN
Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N-methyl-D-aspartic acid (NMDA) receptor class. Development of these ligands seems to be a difficult task because of the conserved region in the binding site of the NMDA receptor subunits. A few scaffolds have been developed showing potential to differentiate between the NMDA receptors.
Asunto(s)
Agonistas de Aminoácidos Excitadores/química , Glutamatos/química , Receptores de Glutamato/química , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Sitios de Unión , Cristalografía por Rayos X , Glutamatos/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
On-column stopped flow multidimensional HPLC (sfMDHPLC) and dynamic high-performance liquid chromatography were applied to investigate the influence of alkyl substituents at the sulfonamidic and amino moieties of benzothiadiazine 1,1-dioxide derivatives on hydrolysis and enantiomerization rate constants. The data obtained indicate the presence of pyrrolo substituent at the 3,4 positions on benzothiadiazine rings inhibits the hydrolysis, whereas the enantiomerization occurs in acidic medium. Hydrolysis rates are quite similar for the two benzothiadiazines methyl substituted to nitrogen at 2- and 4-positions. Conversely, enantiomerization rate of 4-N-methyl substituted is significantly higher than 2-N-methyl substituted.
Asunto(s)
Benzotiadiazinas/química , Benzotiadiazinas/aislamiento & purificación , Química Farmacéutica/métodos , Cromatografía/instrumentación , Cromatografía/métodos , Cromatografía Líquida de Alta Presión/métodos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Modelos Químicos , Nitrógeno/química , Preparaciones Farmacéuticas/aislamiento & purificación , Estereoisomerismo , Termodinámica , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/químicaRESUMEN
Glyphosate (GP) is a widely used herbicide worldwide, yet accumulation of GP and its main byproduct, aminomethylphosphonic acid (AMPA), in soil and water has raised concerns about its potential effects on human health. Thermal treatment, in which contaminants are vaporised and decomposed in the gas-phase, is one option for decontaminating material containing GP and AMPA, yet the thermal decomposition chemistry of these compounds remains poorly understood. Here, we have revealed the thermal decomposition mechanism of GP and AMPA in the gas phase by applying computational chemistry and reaction rate theory methods. The preferred decomposition channel for both substances involves the elimination of P(OH)3 to yield the imine N-methylene-glycine (from GP) or methanimine (from AMPA), with relatively low barrier heights (ca. 45 kcal mol-1). The half-life of GP and AMPA at 1000 K are predicted to be 0.1 and 4 ms respectively, and they should be readily destroyed via conventional incineration processes. The further decomposition of N-methylene-glycine is expected to also take place at similar temperatures, leading to N-methyl-methanimine + CO2, with a barrier height of ca. 48 kcal mol-1. The imine decomposition products of GP and AMPA are expected to react with water vapour to form simple amines and carbonyl compounds.
Asunto(s)
Glicina/análogos & derivados , Herbicidas , Aminas , Glicina/química , Herbicidas/química , Isoxazoles/química , Cinética , Tetrazoles/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , GlifosatoRESUMEN
Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. Determining the structural differences between the binding sites of different subtypes is crucial to our understanding of neuronal circuits and to the development of subtype specific drugs. The structures of the binding domain (S1S2) of the GluR3 (flip) AMPA receptor subunit bound to glutamate and AMPA and the GluR2 (flop) subunit bound to glutamate were determined by X-ray crystallography to 1.9, 2.1, and 1.55 A, respectively. Overall, the structure of GluR3 (flip) S1S2 is very similar to GluR2 (flop) S1S2 (backbone RMSD of 0.30 +/- 0.05 for glutamate-bound and 0.26 +/- 0.01 for AMPA-bound). The differences in the flip and flop isoforms are subtle and largely arise from one hydrogen bond across the dimer interface and associated water molecules. Comparison of the binding affinity for various agonists and partial agonists suggest that the S1S2 domains of GluR2 and GluR3 show only small differences in affinity, unlike what is found for the intact receptors (with the exception of one ligand, Cl-HIBO, which has a 10-fold difference in affinity for GluR2 vs. GluR3).
Asunto(s)
Glutamatos/química , Estructura Terciaria de Proteína , Receptores AMPA/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Sitios de Unión , Unión Competitiva , Cristalografía por Rayos X , Dimerización , Glutamatos/metabolismo , Cinética , Modelos Moleculares , Estructura Molecular , Unión Proteica , Ensayo de Unión Radioligante , Receptores AMPA/metabolismo , Tritio , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
We have evaluated the toxicity of four glyphosate (G)-based herbicides in Roundup formulations, from 10(5) times dilutions, on three different human cell types. This dilution level is far below agricultural recommendations and corresponds to low levels of residues in food or feed. The formulations have been compared to G alone and with its main metabolite AMPA or with one known adjuvant of R formulations, POEA. HUVEC primary neonate umbilical cord vein cells have been tested with 293 embryonic kidney and JEG3 placental cell lines. All R formulations cause total cell death within 24 h, through an inhibition of the mitochondrial succinate dehydrogenase activity, and necrosis, by release of cytosolic adenylate kinase measuring membrane damage. They also induce apoptosis via activation of enzymatic caspases 3/7 activity. This is confirmed by characteristic DNA fragmentation, nuclear shrinkage (pyknosis), and nuclear fragmentation (karyorrhexis), which is demonstrated by DAPI in apoptotic round cells. G provokes only apoptosis, and HUVEC are 100 times more sensitive overall at this level. The deleterious effects are not proportional to G concentrations but rather depend on the nature of the adjuvants. AMPA and POEA separately and synergistically damage cell membranes like R but at different concentrations. Their mixtures are generally even more harmful with G. In conclusion, the R adjuvants like POEA change human cell permeability and amplify toxicity induced already by G, through apoptosis and necrosis. The real threshold of G toxicity must take into account the presence of adjuvants but also G metabolism and time-amplified effects or bioaccumulation. This should be discussed when analyzing the in vivo toxic actions of R. This work clearly confirms that the adjuvants in Roundup formulations are not inert. Moreover, the proprietary mixtures available on the market could cause cell damage and even death around residual levels to be expected, especially in food and feed derived from R formulation-treated crops.
Asunto(s)
Apoptosis , Glicina/análogos & derivados , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular , Femenino , Glicina/metabolismo , Glicina/toxicidad , Humanos , Riñón/citología , Riñón/embriología , Necrosis , Placenta/citología , Polietilenglicoles/química , Polietilenglicoles/toxicidad , Células Madre , Succinato Deshidrogenasa/metabolismo , Venas Umbilicales/citología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/toxicidad , GlifosatoRESUMEN
The glutamate receptor system is implicated in the development and maintenance of epileptic seizures and it has been reported that compounds showing high affinity for both AMPA and KA binding sites are more potent anticonvulsants than compounds having selective affinity toward AMPA or KA receptor. These outcomes make such inhibitors future potential antiepileptic drugs. So, the pair wise binding affinity for AMPA and KA receptors inhibition was proposed by using the addition between biological activities of ligands. This approach for evaluation of pair wise binding affinity was exemplified using set of triazolo [1,5-a] quinoxaline for AMPA and KA receptors. The biological activity towards AMPA and KA receptors (expressed as -log IC(5O)) was taken as a dependent variable for building CoMFA and CoMSIA models. The resulting models show the ways of increasing binding affinity to both AMPA and KA receptors as potential target for epilepsy. The statistically significant results show that pair wise CoMFA and CoMSIA models are better then individual models. The resulting cross-validated r(2)(CV) value 0.806 for CoMFA is greater then 0.780 for CoMSIA pair wise model. The non-cross validated run giving a coefficient of determination r(2) value of 0.946 and 0.908 for CoMFA and CoMSIA respectively, provided a good correlation between the observed and computed affinities of the compounds.
Asunto(s)
Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Quinoxalinas/química , Receptores de Ácido Kaínico/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sitios de Unión , Concentración 50 Inhibidora , Estructura Molecular , Unión Proteica , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/químicaRESUMEN
Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with the extent of domain separation decreasing in the order of apo > DNQX > kainate > glutamate approximately equal to AMPA. These results suggest that agonist-induced domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also promote a 180 degrees flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in the intact receptor and mechanisms by which allosteric effectors modulate receptor activity.
Asunto(s)
Modelos Moleculares , Conformación Proteica , Receptores AMPA/química , Sitios de Unión/genética , Sitios de Unión/fisiología , Cristalografía por Rayos X , Dimerización , Agonistas de Aminoácidos Excitadores/química , Antagonistas de Aminoácidos Excitadores/química , Ácido Glutámico/química , Ácido Kaínico/química , Ligandos , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína/genética , Quinoxalinas/química , Receptores AMPA/agonistas , Receptores AMPA/antagonistas & inhibidores , Relación Estructura-Actividad , Zinc/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/químicaRESUMEN
The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) class of ionotropic glutamate receptors comprises four different subunits: iGluR1/iGluR2 and iGluR3/iGluR4 forming two subgroups. Three-dimensional structures have been reported only of the ligand-binding core of iGluR2. Here, we present two X-ray structures of a soluble construct of the R/G unedited flip splice variant of the ligand-binding core of iGluR4 (iGluR4(i)(R)-S1S2) in complex with glutamate or AMPA. Subtle, but important differences are found in the ligand-binding cavity between the two AMPA receptor subgroups at position 724 (Tyr in iGluR1/iGluR2 and Phe in iGluR3/iGluR4), which in iGluR4 may lead to displacement of a water molecule and hence points to the possibility to make subgroup specific ligands.
Asunto(s)
Ácido Glutámico/química , Receptores AMPA/agonistas , Receptores AMPA/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Cristalografía por Rayos X , Ligandos , Conformación Proteica , Receptores AMPA/genéticaRESUMEN
This paper reports the synthesis and AMPA, Gly/NMDA, and KA receptor binding affinities of a new set of 1,9-disubstituted-8-chloro-pyrazolo[1,5-c]quinazoline-2-carboxylates 2-34. Binding data show that, in general, compounds 2-34 bind to the AMPA receptor with good affinity and selectivity. In particular, the obtained results indicate that the contemporary presence of a 1,2-dicarboxylic acid moiety and suitable benzo-substituents on the PQZ system is important to gain selective AMPA receptor antagonists. Moreover, this study shows that the presence of a 2-carboxybenzoylamino substituent at position-9 (compounds 33-34) is important for obtaining selective KA receptor antagonists. Some selected compounds were also tested for their functional antagonistic activity at both AMPA and NMDA receptor-ion channels.
Asunto(s)
Pirazoles/química , Quinazolinas/síntesis química , Quinazolinas/farmacología , Receptores de Ácido Kaínico/antagonistas & inhibidores , Receptores de Ácido Kaínico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/síntesis química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Estructura Molecular , N-Metilaspartato/química , Quinazolinas/química , Relación Estructura-Actividad , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/químicaRESUMEN
Taking into account structure-activity relationships obtained with our previous series, new diversely substituted 1,2,4-pyridothiadiazine 1,1-dioxides were designed to obtain novel AMPA potentiators. The aim of this work was focused on the improvement of lipophilicity, which is well known as a critical parameter to obtain in vivo active central nervous system agents. For this purpose, two positions on the pyridine ring were privileged to insert selected groups. Among the synthesized compounds emerged 7-chloro-4-ethyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide (12d), which was evaluated in two memory tests in Wistar rats and showed cognition enhancing effects after intraperitoneal injection at doses as low as 0.3mg/kg.
Asunto(s)
Diazóxido/farmacología , Receptores AMPA/efectos de los fármacos , Tiadiazinas/síntesis química , Tiadiazinas/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Animales , Diazóxido/química , Oocitos/efectos de los fármacos , Oocitos/fisiología , Ratas , Ratas Wistar , Receptores AMPA/biosíntesis , Receptores AMPA/genética , Solubilidad , Relación Estructura-Actividad , Tiadiazinas/química , Xenopus laevis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/químicaRESUMEN
Glutamate is the major excitatory neurotransmitter in the brain. Among the cognate ionotropic glutamate receptors, the subfamily selective for AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) is responsible for most fast excitatory synaptic signaling and plays key roles in synaptic plasticity. AMPA receptors (AMPA-Rs) have also been implicated in a number of neurological disorders. To investigate subunit-specific differences in the ligand binding and activation of AMPA-Rs, the GluR4 AMPA-R ligand-binding domain (LBD) was crystallized in complex with full and partial agonists. This is the first non-GluR2 AMPA-R LBD available for structural analysis. Standard cryoprotection protocols yielded high-resolution diffraction from flash-cooled crystals of the complex with the full agonist glutamate. However, for cocrystals with the partial agonist kainate, systematic screening and optimization of cryoprotection conditions yielded at best mosaic, weak diffraction at 100 K. In contrast, room-temperature data collection from capillary-mounted kainate cocrystals exhibited reproducible diffraction to better than 3 A resolution. Together, these crystals lay the foundation for a structural comparison of LBD-agonist interactions in distinct AMPA-R subunits.
Asunto(s)
Frío , Receptores AMPA/química , Receptores AMPA/aislamiento & purificación , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Animales , Clonación Molecular , Cristalización , Calor , Ligandos , Unión Proteica/fisiología , Conformación Proteica , Estructura Terciaria de Proteína , Ratas , Receptores AMPA/agonistas , Receptores AMPA/fisiología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/químicaRESUMEN
The fate of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) was studied in soil. Labeled glyphosate was used to be able to distinguish the measured quantities of glyphosate and AMPA from the background values since the soil was sampled in a field where glyphosate had been used formerly. After addition of labeled glyphosate, the disappearance of glyphosate and the formation and disappearance of AMPA were monitored. The resulting curves were fitted according to a new EU guideline. The best fit of the glyphosate degradation data was obtained using a first-order multi compartment (FOMC) model. DT(50) values of 9 days (glyphosate) and 32 days (AMPA) indicated relatively rapid degradation. After an aging period of 6 months, the leaching risk of each residue was determined by treating the soil with pure water or a phosphate solution (pH 6), to simulate rain over a non-fertilized or fertilized field, respectively. Significantly larger (p < 0.05) amounts of aged glyphosate and AMPA were extracted from the soil when phosphate solution was used as an extraction agent, compared with pure water. This indicates that the risk of leaching of aged glyphosate and AMPA residues from soil is greater in fertilized soil. The blank soil, to which 252 g glyphosate/ha was applied 21 months before this study, contained 0.81 ng glyphosate/g dry soil and 10.46 ng AMPA/g dry soil at the start of the study. Blank soil samples were used as controls without glyphosate addition. After incubation of the blank soil samples for 6 months, a significantly larger amount of AMPA was extracted from the soil treated with phosphate solution than from that treated with pure water. To determine the degree of uptake of aged glyphosate residues by crops growing in the soil, (14)C-labeled glyphosate was applied to soil 6.5 months prior to sowing rape and barley seeds. After 41 days, 0.006 +/- 0.002% and 0.005 +/- 0.001% of the applied radioactivity was measured in rape and barley, respectively.
Asunto(s)
Monitoreo del Ambiente , Agonistas de Aminoácidos Excitadores/análisis , Glicina/análogos & derivados , Herbicidas/análisis , Contaminantes del Suelo/análisis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/análisis , Agricultura , Radioisótopos de Carbono , Agonistas de Aminoácidos Excitadores/química , Glicina/análisis , Glicina/química , Herbicidas/química , Humanos , Marcaje Isotópico , Medición de Riesgo , Contaminantes del Suelo/química , Factores de Tiempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , GlifosatoRESUMEN
The broad-spectrum herbicide glyphosate is one of the most widely used pesticides. Both glyphosate and its major metabolite, aminomethylphosphonic acid (AMPA), persist in waters; thus, their environmental fates are of interest. We investigated the influence of compost dose, sampling depth, moisture and saturated hydraulic conductivity (Ks) on the persistence of these substances. The amounts of AMPA quantified by triple quadrupole liquid chromatography-mass spectrometry (LC-QqQ-MS/MS) using isotopically labeled extraction standards were higher than those of glyphosate and differed among the samples. Both glyphosate and AMPA showed gradually decreasing concentrations with soil depth, and bootstrapped ANOVA showed significant differences between the contents of glyphosate and AMPA and their behavior related to different compost dosages and sampling depths. However, the compost dose alone did not cause significant differences among samples. Bayesian statistics revealed that the amounts of glyphosate and AMPA were both dependent on the sampling depth and compost dose, but differences were found when considering the physical factors of Ks and moisture. Glyphosate was influenced by moisture but not Ks, whereas AMPA was influenced by Ks but not moisture. Importantly, we found behavioral differences between glyphosate and its major metabolite, AMPA, related to the physical properties of Ks and moisture.
Asunto(s)
Compostaje/métodos , Glicina/análogos & derivados , Contaminantes del Suelo/química , Suelo/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Glicina/química , Contaminantes del Suelo/análisis , Espectrometría de Masas en Tándem/métodos , GlifosatoRESUMEN
Starting from 1-4 and 7 structural templates, analogues based on bioisosteric replacements (5a-c vs 1, 2 and 6 vs 7) were synthesized for completing the SAR analysis. Interesting binding properties at GluA2, GluK1, and GluK3 receptors were discovered. The requirements for GluK3 interaction were elucidated by determining the X-ray structures of the GluK3-LBD with 2 and 5c and by computational studies. Antinociceptive potential was demonstrated for GluK1 partial agonist 3 and antagonist 7 (2 mg/kg ip).
Asunto(s)
Receptores de Ácido Kaínico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/análogos & derivados , Analgésicos/química , Animales , Cristalografía por Rayos X , Ligandos , Unión Proteica , Receptores AMPA , Receptores de Ácido Kaínico/agonistas , Receptores de Ácido Kaínico/antagonistas & inhibidores , Receptores de Ácido Kaínico/metabolismo , Relación Estructura-Actividad , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Receptor Kainato GluK3RESUMEN
Glyphosate (N-(phosphonomethyl)glycine) is a broad-spectrum systemic herbicide used to kill weeds that compete with commercial crops. In Argentina, the use of glyphosate-based herbicides increased dramatically (up to â¼200,000 tons on 2012) since the introduction of glyphosate-resistant crops, such as transgenic soy and resistant corn, and the adoption of non-till practices in the 1990's. Sallow lakes within the Pampa region may be potentially impacted by continuous herbicide usage. We surveyed 52 shallow lakes from the Pampa region (Buenos Aires Province, Argentina) to assess the occurrence and concentrations of glyphosate and its main degradation product (AMPA). For comparison, we also sampled 24 shallow lakes from an area with no agricultural use of glyphosate (Northern Patagonia). Glyphosate and AMPA were analyzed by UPLC-MS/MS ESI (±) in lake water, suspended particulate matter (SPM), and sediment samples. Within the Pampa region, glyphosate residues were detected in >40% of samples. Glyphosate residues were detected more frequently in sediment and surface water than in SPM samples. The mean (maximum) concentrations of glyphosate were 2.11 (4.52) µg l-1 for surface water; 0.10 (0.13) µg l-1 for SPM and 10.47 (20.34) µg kg-1 for sediment samples, respectively. Whereas, mean (maximum) concentrations of AMPA were 0.84 and (0.90) µg l-1 for surface water; 0.07 (0.07) µg l-1 for SPM; and 22.53 (32.89) µg kg-1 for sediment samples. The herbicide was not detected in samples from the Patagonian region. To our knowledge, this is the first study reporting the occurrence and concentrations of the herbicide in freshwater lakes of Argentina.