Métodos de preparação e atividade biológica do ácido quinolínico e derivados

In 1981 2,3-pyridine dicarboxylic acid (quinolinic acid) was discovery to be a selective agonist for the N-methyl -D-aspartic acid (NMDA) receptor. As a consequence it possesses neurotoxic activity resulting from overstimulation of the receptor. Quinolinic acid is implicated as an etiological factor in a range of neurodegenerative disease including AIDS related dementia, Huntington´s disease and Lyme disease. In the design of novel therapies to treat these diseases, some molecules have been identified as an important target. In this paper we described different methods to prepare quinolinic acid and derivatives.

Excitatory amino acid receptor blockade within the caudal pressor area and rostral ventrolateral medulla alters cardiovascular responses to nucleus raphe obscurus stimulation in rats

Pressor responses elicited by stimulation of the nucleus raphe obscurus (NRO) depend on the integrity of the rostral ventrolateral medulla (RVLM). Therefore, to test the participation of excitatory amino acid (EAA) receptors in the cardiovascular responses evoked by NRO stimulation (1 ms, 100 Hz, 40-70 µA, for 10 s), the EAA antagonist kynurenic acid (Kyn) was microinjected at different sites in the ventrolateral medullar surface (2.7 nmol/200 nl) of male Wistar rats (270-320 g, N = 39) and NRO stimulation was repeated. The effects of NRO stimulation were: hypertension (deltaMAP = +43 ± 1 mmHg, P<0.01), bradycardia (deltaHR = -30 ± 7 bpm, P<0.01) and apnea. Bilateral microinjection of Kyn into the RVLM, which did not change baseline parameters, almost abolished the bradycardia induced by NRO stimulation (deltaHR = -61 ± 3 before vs -2 ± 3 bpm after Kyn, P<0.01, N = 7). Unilateral microinjection of Kyn into the CVLM did not change baseline parameters or reduce the pressor response to NRO stimulation (deltaMAP = +46 ± 5 before vs +48 ± 5 mmHg after Kyn, N = 6). Kyn bilaterally microinjected into the caudal pressor area reduced blood pressure and heart rate and almost abolished the pressor response to NRO stimulation (deltaMAP = +46 ± 4 mmHg before vs +4 ± 2 mmHg after Kyn, P<0.01, N = 7). These results indicate that EAA receptors on the medullary ventrolateral surface play a role in the modulation of the cardiovascular responses induced by NRO stimulation, and also suggest that the RVLM participates in the modulation of heart rate responses and that the caudal pressor area modulates the pressor response following NRO stimulation.

Protective mechanism of agmatine pretreatment on RGC-5 cells injured by oxidative stress

Agmatine has neuroprotective effects on retinal ganglion cells (RGCs) as well as cortical and spinal neurons. It protects RGCs from oxidative stress even when it is not present at the time of injury. As agmatine has high affinity for various cellular receptors, we assessed protective mechanisms of agmatine using transformed RGCs (RGC-5 cell line). Differentiated RGC-5 cells were pretreated with 100 μM agmatine and consecutively exposed to 1.0 mM hydrogen peroxide (H2O2). Cell viability was determined by measuring lactate dehydrogenase (LDH), and the effects of selective alpha 2-adrenergic receptor antagonist yohimbine (0-500 nM) and N-methyl-D-aspartic acid (NMDA) receptor agonist NMDA (0-100 µM) were evaluated. Agmatine’s protective effect was compared to a selective NMDA receptor antagonist MK-801. After a 16-h exposure to H2O2, the LDH assay showed cell loss greater than 50%, which was reduced to about 30% when agmatine was pretreated before injury. Yohimbine almost completely inhibited agmatine’s protective effect, but NMDA did not. In addition, MK-801 (0-100 µM) did not significantly attenuate the H2O2-induced cytotoxicity. Our results suggest that neuroprotective effects of agmatine on RGCs under oxidative stress may be mainly attributed to the alpha 2-adrenergic receptor signaling pathway.

The structure of arabidopsis NPR1 : its function as a salicylic acid receptor and a metal-binding protein

The Arabidopsis NPRI protein regulates systemic acquired resistance dependent on salicylic acid. Analyses by plant two-hybrid analysis in vivo and pull-down assays in vitro showed that the BTB/POZ domain of NPRI at the N-terminus serves as an autoinhibitory domain to negate the function of the transactivation domain at the C-terminus through direct binding of these two domains. I t was also shown that the binding of the BTB/POZ domain to the C-terminus of NPRI was abolished by SA treatment, suggesting that SA could interfere directly with this binding. By gel filtration, it was demonstrated that SA affects the conformation of full-length NPRl , confirming the role of NPRI as an SA receptor. Gel filtration analysis also indicated that NPRI could be converted from an oligomer to a dimer with SA treatment. Furthermore, one N-terminal deletion ~513 has been shown to act as a metal-binding protein and its two Cys-521 and Cys-529 are important for binding to Ni 2 + by pull-down assays.

Enhancement Of High Affinity Y-Aminobutyric Acid Receptor Binding In Cerebellum Of Pyridoxine-Deficient Rat

The high-affinity of [3H]y-aminobutyric acid (GABA) to GABAA receptors and [3H]baclofen to GABAB receptors were studied in the cerebellum of pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding ( Bmax) of both GABAA and GABAB receptors with no significant difference in their binding affinities (Kd). The changes observed suggest a supersensitivity of GABAA and GABAB receptors which seems to correlate negatively with the concentration of GABA in the cerebellum of pyridoxine-deficient rats.

Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system

BACKGROUND: Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract. METHODS: We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility. KEY RESULTS: The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to approximately 50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit. CONCLUSIONS & INFERENCES: G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.

Potential tuberculostatic agents: Micelle-forming copolymer poly(ethylene glycol)-poly(aspartic acid) prodrug with isoniazid

With the objective of obtaining slow-acting isoniazid derivatives, of potential use as chemoprophylactics or chemotherapeutics in tuberculosis, the micelle-forming copolymer of poly(ethylene glycol)-poly(aspartic acid) prodrug with isoniazid was synthesized. The derivative obtained was found to be active in Mycobacterium Il(tuberculosis culture, with a minimal inhibitory concentration (MIC) 5.6 times lower than that of the tuberculostatic drug.

Computer-aided design of a novel ligand for retinoic acid receptor in cancer chemotherapy

The isotypes of RAR and RXR are retinoic acid and retinoid X acid receptors, respectively, whose ligand-binding domain contains the ligand-dependent activation function, with distinct pharmacological targets for retinoids, involved in the treatment of various cancers and skin diseases. Due to the major challenge which cancer treatment and cure still imposes after many decades to the international scientific community, there is actually considerable interest in new ligands with increased bioactivity. We have focused on the retinoid acid receptor, which is considered an interesting target for drug design. In this work, we carried out density functional geometry optimizations, and different docking procedures. We performed screening in a large database (hundreds of thousands of molecules which we optimized at the AM1 level) yielding a set of potential bioactive ligands. A new ligand was selected and optimized at the B3LYP/6-31G* level. A flexible docking program was used to investigate the interactions between the receptor and the new ligand. The result of this work is compared with several crystallographic ligands of RAR. Our theoretically more bioactive new-ligand indicates stronger and more hydrogen bonds as well as hydrophobic interactions with the receptor. (c) 2005 Wiley Periodicals, Inc.

Fluorescence study of the interaction between metal ions and methyl methacrylate methacrylic acid copolymers in aqueous solutions: thallium(I), calcium(II), and terbium(III)

The binding of the cations thallium(I), calcium(II) and terbium(III) to methyl methacrylate-methacrylic acid copolymers with different fractions of acid groups (x) has been studied in aqueous solution at, various pH values using the fluorescence of covalently bonded 9-vinyl anthracene as a probe. In all cases, the extent of binding increases as a function of the charge of the polymer with either increasing fraction of carboxylic acids or of pH. However, differences are observed in the behavior of the three cations, With Tl(I), quenching of the anthracene group fluorescence is observed. indicating that the thallium(I) approaches the probe and suggesting that the alkylanthracene is probably in a relatively polar region. Binding constants have been determined from anthracene quenching data and from studies with the fluorescent-probe sodium pyrenetetrasulfonate, Good agreement is obtained between the two methods, and values for the binding constants increase from 250 to 950 M-1 as x increases from 0.39 to 1. It is suggested that the cation is held in the polyelectrolyte domain, partly by Debye-Huckel effects and partly by more specific interactions. Stronger binding is found with calcium(II) and terbium(III), and in this case increases in fluorescence intensity are observed on complexation due to the anthracene group being in a more hydrophobic region, probably as a result of conformational changes in the polymer chain. In the former case the stoichiometry of the interaction was determined from the fluorescence data to involve two carboxylate groups bound per calcium. Association constants were found using murexide as an indicator of free calcium to vary from 8400 to 37 000 M-1 as x increases from 0.39 to 1. It is suggested that in this case specific calcium(II)-carboxylate interactions contribute to the binding. With terbium(III), a greater increase in the probe fluorescence intensity was observed than with calcium, and it is suggested that the interaction with the polymer is even stronger, leading to a more pronounced conformational change in the polymer. It is proposed that the terbium(III) interacts with sis carboxylic groups on the polymer chain, with three being coordinated and three attracted by electrostatic interactions.

Aspartic acid racemization dating of Holocene brachiopods and bivalves from the southern Brazilian shelf, South Atlantic

The extent of racemization of aspartic acid (Asp) has been used to estimate the ages of 9 shells of the epifaunal calcitic brachiopod Bouchardia rosea and 9 shells of the infaunal aragonitic bivalve Semele casali. Both taxa were collected concurrently from the same sites at depths of 10 m and 30 m off the coast of Brazil. Asp D/L values show an excellent correlation with radiocarbon age at both sites and for both taxa (r(Site)(2) (9) (B. rosea) = 0.97 r(Site)(2) (1) (B.) (rosea) = 0.997, r(Site)(2) (9) (S.) (casali) = 0.9998, r(2) (Site) (1) (S.casali) = 0.93). The Asp ratios plotted against reservoir-corrected AMS radiocarbon ages over the time span of multiple millennia can thus be used to develop reliable and precise geochronologies not only for aragonitic mollusks (widely used for dating previously), but also for calcitic brachiopods. At each collection site, Bouchardia specimens display consistently higher D/L values than specimens of Semele. Thermal differences between sites are also notable and in agreement with theoretical expectations, as extents of racemization for both taxa are greater at the warmer, shallower site than at the cooler, deeper one. In late Holocene marine settings, concurrent time series of aragonitic and calcitic shells can be assembled using Asp racemization dating, and parallel multi-centennial to multi-millennial records can be developed simultaneously for multiple biomineral systems. (c) 2006 University of Washington. All rights reserved.

Citral: A monoterpene with prophylactic and therapeutic anti-nociceptive effects in experimental models of acute and chronic pain

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Association of retinoic acid receptor genes with meningomyelocele.

BACKGROUND: Neural tube defects (NTDs) occur in as many as 0.5-2 per 1000 live births in the United States. One of the most common and severe neural tube defects is meningomyelocele (MM) resulting from failed closure of the caudal end of the neural tube. MM has been induced by retinoic acid teratogenicity in rodent models. We hypothesized that genetic variants influencing retinoic acid (RA) induction via retinoic acid receptors (RARs) may be associated with risk for MM. METHODS: We analyzed 47 single nucleotide polymorphisms (SNPs) that span across the three retinoic acid receptor genes using the SNPlex genotyping platform. Our cohort consisted of 610 MM families. RESULTS: One variant in the RARA gene (rs12051734), three variants in the RARB gene (rs6799734, rs12630816, rs17016462), and a single variant in the RARG gene (rs3741434) were found to be statistically significant at p < 0.05. CONCLUSION: RAR genes were associated with risk for MM. For all associated SNPs, the rare allele conferred a protective effect for MM susceptibility.

Analysis of endogenous retinoic acid receptor expression and function in murine melanoma cells

There have been numerous reports over the past several years on the ability of vitamin A analogs (retinoids) to modulate cell proliferation, malignant transformation, morphogenesis, and differentiation in a wide variety of cell types and organisms. Two families of nuclear retinoid-inducible, trans-acting, transcription-enhancing receptors that bear strong DNA sequence homology to thyroid and steroid hormone receptors have recently been discovered. The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) each have at least three types designated $\alpha,$ $\beta,$ and $\gamma,$ which are encoded by separate genes and expressed in a tissue and cell type-specific manner. We have been interested in the mechanism by which retinoids inhibit tumor cell proliferation and induce differentiation. As a model system we have employed several murine melanoma cell lines (S91-C2, K1735P, and B16-F1), which are sensitive to the growth-inhibitory and differentiation-inducing effects of RA, as well as a RA-resistant subclone of one of the cell lines (S91-C154), in order to study the role of the nuclear RARs in these effects. The initial phase of this project consisted of the characterization of the expression pattern of the three known RAR and RXR types in the murine melanoma cell lines in order to determine whether any differences exist which may elucidate a role for any of the receptors in RA-induced growth inhibition and differentiation. The novel finding was made that the RAR-$\beta$ gene is rapidly induced from undetectable levels by RA treatment at the mRNA and protein level, and that the induction of RAR-$\beta$ by other biologically active retinoids correlated with their ability to inhibit the growth of the highly RA-sensitive S91-C2 cell line. This suggests a role for RAR-$\beta$ in the growth inhibiting effect of retinoids. The second phase of this project involves the stable expression of RAR-$\beta$ in the S91-C2 cells and the RAR-$\beta$ receptor-null cell line, K1735P. These studies have indicated an inverse correlation between RAR-$\beta$ expression and proliferation rate. ^