Enantiomeric N-substituted phthalimides with excitatory amino acids protect zebrafish larvae against PTZ-induced seizures.

Epilepsy is a chronic neurological disease with high prevalence and adverse impacts on the quality of life of patients and caregivers. Up to one-third of individuals with epilepsy do not respond to current pharmacotherapy, underscoring the importance of identifying new molecules for epilepsy control. Thalidomide, the first synthetized phthalimide, is a neuroactive molecule with anti-seizure drug properties. The phthalimide group has been studied in some N-phthaloyl amino acids due to its pharmacological properties. Here we examine enantiomers of phthaloyl aspartate (R and S) and phthaloyl glutamate (R and S) for anti-seizure effects using zebrafish as a model. The zebrafish model is rapidly growing in use as a preclinical screening tool for drug discovery in epilepsy. Pentylenetetrazol (PTZ) exposure was used to produce convulsive behavior in 7- and 10-days post-fertilization (dpf) zebrafish larvae; these ages correspond to before and after the blood-brain-barrier (BBB) is fully developed. Larvae were pre-treated for 60 min with: control, valproic acid sodium salt (SVP) 3 mM, or one of two concentrations of N-phthaloyl-R-glutamic acid (R-TGLU; 100, 316 µM) prior to PTZ addition. R-TGLU modified the locomotor phenotype and protected against PTZ in 7 and 10 dpf larvae at 316 µM, suggesting it crossed the BBB. We next tested the per se and anticonvulsant effect of the glutamate and aspartate phthalimides were tested at 237.1 and 316 µM concentration in 10dpf zebrafish. The four tested molecules produced an anticonvulsant effect at 237.1 µM concentration, however the behavioral changes that they induce suggest that they might act by different mechanisms.

Marine Excitatory Amino Acids: Structure, Properties, Biosynthesis and Recent Approaches to Their Syntheses.

This review considers the results of recent studies on marine excitatory amino acids, including kainic acid, domoic acid, dysiherbaine, and neodysiherbaine A, known as potent agonists of one of subtypes of glutamate receptors, the so-called kainate receptors. Novel information, particularly concerning biosynthesis, environmental roles, biological action, and syntheses of these marine metabolites, obtained mainly in last 10-15 years, is summarized. The goal of the review was not only to discuss recently obtained data, but also to provide a brief introduction to the field of marine excitatory amino acid research.

Chiral analysis of amino acid neurotransmitters and neuromodulators in mouse brain by CE-LIF.

Chiral CE method has been developed for quantitative determination of d-amino acid modulators of NMDA glutamate receptor; d-serine and d-aspartate along with l-glutamate and l-aspartate in biological samples. These ligands are suggested to be involved in regulation of NMDA receptor related brain functions, such as neurogenesis, neuronal plasticity, and memory formation. For sensitive determination of the amino acids LIF detection was chosen, and a fluorogenic reagent, 7-fluoro-4-nitro-2,1,3-benzoxadiazole was used for derivatization. An amino-modified ß-CD, 6-monodeoxy-6-mono(3-hydroxy)propylamino-ß-CD (HPA-ß-CD) was applied as chiral selector. Determinations were accomplished in a polyacrylamide coated capillary and reverse polarity was used for the analysis of the negatively charged analytes. The method was optimized and validated; 6 mM HPA-ß-CD in 50 mM HEPES buffer, pH 7 was appropriate to achieve baseline separation of the analytes. The limit of quantification with acceptable accuracy is 0.05 µM for both d-amino acids. The method was used for the determination of d-aspartate and d-serine content in various brain regions of adult mice.

An optimized HPLC/MS/MS method for quantification of excitatory amino acids in rat hippocampus and its application in brain ischemia/reperfusion research.

An optimized HPLC/MS/MS method was established to quantify glutamate (Glu) and aspartic acid (Asp) in rat hippocampus with glutamate-d5 (Glu-d5) as internal standard. The mass spectrometry was operated under the multiple reaction monitoring mode using electrospray ionization in the positive ion mode for Glu and negative ion mode for Asp. The retention times of Glu, Asp and Glu-d5 were 1.53, 2.07 and 1.52 min, respectively. The linearity of calibration curves was good, with r(2) > 0.99 and a lower limit of quantitation of 10 ng/mL. The intra-day precisions (relative standard deviation, RSD) of Glu and Asp were in the range of 3.61-8.17 and 4.22-10.09%, respectively; the inter-day precisions (RSD) of Glu and Asp were in the range of 3.57-5.19 and 2.49-5.04%, respectively. The accuracies of Glu and Asp were in the range of -2.10-6.20 and -0.90-10.00%, respectively. The recovery rates of 10, 100 and 1000 ng/mL were found to be 0.89 ± 0.24, 1.01 ± 0.10 and 0.90 ± 0.12 for Glu and 0.99 ± 0.26, 0.93 ± 0.07 and 1.13 ± 0.13 for Asp, respectively. This optimized method was successfully applied to quantify the concentration of Glu and Asp in rat hippocampus in brain ischemia/reperfusion research.

Chiral separation and determination of excitatory amino acids in brain samples by CE-LIF using dual cyclodextrin system.

Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of D-Asp in the central nervous system. To achieve appropriate detection sensitivity fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and laser-induced fluorescence detection was applied. Although, simultaneous baseline separation of the two enantiomer pairs could be achieved by using 3 mM 6-monodeoxy-6-mono(3-hydroxy)propylamino-ß-cyclodextrin (HPA-ß-CD), further improvement of the chemical selectivity was required because of the high excess of L-enantiomers in real samples to be analyzed. The system selectivity was fine-tuned by combination of 8 mM heptakis(2,6-di-O-methyl)-ß-cyclodextrin and 5 mM HPA-ß-CD in order to increase the resolution between aspartate and glutamate enantiomers. The method was validated for biological application. The limits of detection for D-Asp and D-Glu were 17 and 9 nM, respectively, while the limit of quantification for both analytes was 50 nM. This is the lowest quantification limit reported so far for NBD-tagged D-Asp and D-Glu obtained by validated capillary electrophoresis laser-induced fluorescence method. The applicability of the method was demonstrated by analyzing brain samples of 1-day-old chickens. In all the studied brain areas, the D-enantiomer contributed 1-2 % of the total aspartate content, corresponding to 17-45 nmol/g wet tissue.

Rare excitatory amino acid from flowers of zonal geranium responsible for paralyzing the Japanese beetle.

The Japanese beetle (JB), Popillia japonica, exhibits rapid paralysis after consuming flower petals of zonal geranium, Pelargonium x hortorum. Activity-guided fractionations were conducted with polar flower petal extracts from P. x hortorum cv. Nittany Lion Red, which led to the isolation of a paralysis-inducing compound. High-resolution-MS and NMR ((1)H, (13)C, COSY, heteronuclear sequential quantum correlation, heteronuclear multiple bond correlation) analysis identified the paralytic compound as quisqualic acid (C(5)H(7)N(3)O(5)), a known but rare agonist of excitatory amino acid receptors. Optical rotation measurements and chiral HPLC analysis determined an L-configuration. Geranium-derived and synthetic L-quisqualic acid demonstrated the same positive paralytic dose-response. Isolation of a neurotoxic, excitatory amino acid from zonal geranium establishes the phytochemical basis for induced paralysis of the JB, which had remained uncharacterized since the phenomenon was first described in 1920.

L-aspartate as an amino acid neurotransmitter: mechanisms of the depolarization-induced release from cerebrocortical synaptosomes.

The role of L-aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well known excitatory neurotransmitter L-glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca2+. At 35 and 50 mM KCl, the overflows of L-aspartate, but not those of L-glutamate, became sensitive to DL-threo-b-benzyloxy aspartic acid (DL-TBOA), an excitatory amino acid transporter inhibitor. In the presence of DL-TBOA, the 50 mM KCl-evoked release of L-aspartate was still largely external Ca2+-dependent. The DL-TBOA insensitive,external Ca2+-independent component of the 50 mM KCl-evoked overflows of L-aspartate and L-glutamate was significantly decreased by the mitochondrial Na+/Ca2+ exchanger blocker CGP 37157. The Ca2+-dependent, KCl-evoked overflows of L-aspartate and L-glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-induced L-aspartate and L-glutamate release was completely external Ca2+-dependent and never affected by DL-TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [3H]D aspartate and [3H]L-glutamate. Therefore, our data showing that L-aspartate is released from nerve terminals by calcium dependent,exocytotic mechanisms support the neurotransmitter role of this amino acid.

[Native and chiral modified NMDA-receptor NR1-binding core ligands modeling].

Native and chiral modified (with non-enzymatic Asn racemization) NR1-binding core of NMDA-receptor was modelled by means of molecular dynamic ligand modelling. It was concluded that Gly, D-Ser, D-Asn and D-Thr are ligands of NR1-binding core native NMDA-receptor, whereas the chiral modified NR1-binding core is characterized by the aliphatic non-polar amino acids D-Ala, D-Leu, D-Ile and D-Pro as ligands. The latter amino acids can be considered as effective ligands of NMDA-receptor NR1-binding core in age-related pathology.

The influence of CRF and alpha-helical CRF(9-41) on rat fear responses, c-Fos and CRF expression, and concentration of amino acids in brain structures.

In the present study we have examined the influence of intracerebroventricullary administered CRF, and a non-selective CRF receptor antagonist, alpha-helical CRF((9-41)), on rat conditioned fear response, serum corticosterone, c-Fos and CRF expression, and concentration of amino acids (in vitro), in several brain structures. Pretreatment of rats with CRF in a dose of 1 microg/rat, enhanced rat-freezing response, and further increased conditioned fear-elevated concentration of serum corticosterone. Moreover, exogenous CRF increased aversive context-induced expression of c-Fos in the parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN), CA1 area of the hippocampus, and M1 area of the frontal cortex. A different pattern of behavioral and biochemical changes was present after pre-test administration of alpha-helical CRF((9-41)) (10 microg/rat): a decrease in rat fear response and serum corticosterone concentration; an attenuation of fear-induced c-Fos expression in the dentate gyrus, CA1, Cg1, Cg2, and M1 areas of the frontal cortex; a complete reversal of the rise in the number of CRF immunoreactive complexes in the M2 cortical area, induced by conditioned fear. Moreover, alpha-helical CRF((9-41)) increased the concentration of GABA in the amygdala of fear-conditioned rats. Altogether, the present data confirm and extend previous data on the integrative role of CRF in the central, anxiety-related, behavioral and biochemical processes. The obtained results underline also the role of frontal cortex and amygdala in mediating the effects of CRF on the conditioned fear response.

Cellular origin of dysiherbaine, an excitatory amino acid derived from a marine sponge.

The cellular origin of dysiherbaine, a marine-sponge toxin, was investigated immunohistochemically by using an anti-dysiherbaine antibody. Dysiherbaine-like immunoreactivity was found to be localized in spherical cells harbored in the sponge mesohyl. A combination of ribosomal RNA gene (rDNA) analysis and cell-morphology analysis revealed that the spherical cells were Synechocystis cyanobacteria. However, the sponge, identified as Lendenfeldia chondrodes on the basis of its rDNA sequence, appeared to contain two different chemotypes--dysiherbaine-producing (DH+) and nondysiherbaine-producing (DH-)--both of which inhabited the same region. Synechocystis cells in the DH- sponge were not labeled with antibody, although the 16S rDNA gene profile of the cyanobacteria in the DH- sponge was indistinguishable from that of the cyanobacteria in the DH+ sponge. On the basis of these results, we hypothesize that dysiherbaine is a metabolite of certain varieties of endosymbiotic Synechocystis sp.

Analysis of neuroactive amino acids from microdialysate samples by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method.

A sensitive and rapid reversed-phase high-performance liquid chromatographic method using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and fluorescence detection is reported. By directly derivatizing microdialysate samples with AQC, an automatic and rapid simultaneous measurement of aspartate, serine, glutamate, glycine and histidine was developed. Excellent linearity (r2 > or = 0.998) was achieved for the standard mixture used for the validation experiments. Within-day and between-day precision was less than 6.2%, and the accuracy ranged from 95 to 105.2% in standards. This method is suitable for single run analysis of a high number of small volume microdialysate samples from rat hippocampus. Amino acids from microdialysate samples were quantified with RSD for reproducibility below 2%, and at approximately 0.1% for retention time.

[Saltation behavior in excitation spectra of fluorescent molecules].

Excitation spectra are commonly used to study relationship between molecular structure of fluorescent substances and energy transfer during the fluorescence process. It is generally taken for granted that the excitation spectrum of the sample is equivalent to its absorption spectrum, even a copy of the latter. However, exceptions have been found in many cases. Considering various factors that affect the excitation spectra of solution comprehensively, a model has been established to study the behavior of the excitation spectra. After analyzing the model mathematically, including introducing catastrophe theory, we came into the following conclusions: As far as the topological properties are concerned, the excitation spectra are the same as its absorption spectra, provided the concentration of the substance is below a threshold. However, when the concentration is beyond the threshold, the excitation spectra undergo a series of topological saltation, leading to significant a deviation from the absorption spectra. Comparative studies of both excitation and absorption spectra of naphthalene dissolved in n-hexane confirmed the above hypothesis.

[Anticonvulsive activity of antiepileptic drugs and quantum-chemical modelling of their interaction with GABA(A) receptor]. / Antikonvul'santnaia aktivnost' protivoépilepticheskikh preparatov i kvantovo-khimicheskoe modelirovanie ikh vzaimodeistviia s retseptorom GAMK(A).

The results of experimental analysis of the clinical activity of the antiepileptic drugs' (Phenobarbital, Carbamazepine, Valproic acid, Lamotrigine, Topiramate, Felbamate) widely used in clinic, that was carried out using the standard convulsion test with bicuculline in vivo were compared with characteristics of these drugs' interaction with the key aminoacids of GABA(A) receptor calculated by quantum chemical method (program HyperChem7, semi-empirical method AM1 technique). The correlation between the activity of the drugs in the experiment in vivo and energy of system's interaction of the drugs with aminoacid residue Thr201-Thr202-Gly203- Ala204-Tyr205-Pro206 was found out.

Excitatory amino acids.

Recent developments in the understanding of the molecular function of memory and other CNS-mediated processes have arisen from the multidisciplinary interplay of excitatory amino acid synthesis and medicinal chemistry, X-ray crystallographic structural protein analysis, molecular biology, pharmacology and physiology. This review seeks to place recent synthetic developments of EAA analogues in the wider pharmacological setting, illustrating the need for and importance of these compounds.

Enantioselective synthesis of the excitatory amino acid (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid.

An enantioselective synthesis of the alpha,alpha-dialkyl-alpha-amino acid (1S,3R)-ACPD has been achieved using an alkylidene carbene 1,5-CH insertion reaction as a key step. The ketone cyclization precursor was synthesized from Garner's aldehyde in high yield via a Wittig homologation and subsequent catalytic hydrogenation. Treatment of the ketone with 1.2 equiv of lithio(trimethylsilyl)diazomethane in THF resulted in the formation of the corresponding cyclopentene-containing CH-insertion product in 62-69% yield in high enantiomeric excess. Subsequent functional group manipulation allowed the synthesis of the amino acid (1S,3R)-ACPD to be completed.

A direct chemical interaction between dynorphin and excitatory amino acids.

The endogenous opioid peptide dynorphin A elicits non-opioid receptor-mediated neurotoxic effects. These effects are blocked by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists. Herein, the mechanism for the non-opioid effects of dynorphin and related peptides was studied by matrix-assisted laser desorption ionization (MALDI) mass-spectrometry. We observed that both glutamate or aspartate bind non-covalently to dynorphin A and dynorphin 2-17. However, when dynorphin A or dynorphin 2-17 were added to an equimolar mixture of Glutamate and Aspartate, they both complexed preferentially with glutamate. These data may explain the non-opioid physiological effects of dynorphin A and related peptides and indicate that the direct chemical interaction between neurotransmitters should be monitored when studying interactions between different neurochemical systems.

The cDNA sequence of an excitatory insect selective neurotoxin from the scorpion Buthus martensi Karsch.

The full-length cDNA of an excitatory insect selective neurotoxin was amplified from total cDNAs of venomous glands of the scorpion Buthus martensi Karsch (BmK) using the 3'RACE and 5'RACE (rapid amplification of cDNA ends, RACE) method and sequenced. The cDNA encoded a precursor of the insect toxin of 88 amino acid residues, including a signal peptide of 18 residues and a mature toxin of 70 residues. The cDNA deduced sequence of this toxin was homologous with the determined amino acid sequence of BmK IT1, an excitatory insect toxin purified from the scorpion venom, except for three different residues, two at the positions 24-25, and another in the COOH-terminus of the toxin. Among them the COO-terminal residue Gly in the cDNA deduced sequence was predominantly different from the conserved residue Asn found in other known scorpion excitatory insect toxins.