Unveiling the Venom Composition of the Colombian Coral Snakes Micrurus helleri, M. medemi, and M. sangilensis.

Little is known of the biochemical composition and functional features of the venoms of poorly known Colombian coral snakes. Here, we provide a preliminary characterization of the venom of two Colombian endemic coral snake species, Micrurus medemi and M. sangilensis, as well as Colombian populations of M. helleri. Electrophoresis and RP-HPLC techniques were used to identify venom components, and assays were conducted to detect enzyme activities, including phospholipase A2, hyaluronidase, and protease activities. The median lethal dose was determined using murine models. Cytotoxic activities in primary cultures from hippocampal neurons and cancer cell lines were evaluated. The venom profiles revealed similarities in electrophoretic separation among proteins under 20 kDa. The differences in chromatographic profiles were significant, mainly between the fractions containing medium-/large-sized and hydrophobic proteins; this was corroborated by a proteomic analysis which showed the expected composition of neurotoxins from the PLA2 (~38%) and 3FTx (~17%) families; however, a considerable quantity of metalloproteinases (~12%) was detected. PLA2 activity and protease activity were higher in M. helleri venom according to qualitative and quantitative assays. M. medemi venom had the highest lethality. All venoms decreased cell viability when tested on tumoral cell cultures, and M. helleri venom had the highest activity in neuronal primary culture. These preliminary studies shed light on the venoms of understudied coral snakes and broaden the range of sources that could be used for subsequent investigations of components with applications to specific diseases. Our findings also have implications for the clinical manifestations of snake envenoming and improvements in its medical management.

Antimicrobial Activity Developed by Scorpion Venoms and Its Peptide Component.

Microbial infections represent a problem of great importance at the public health level, with a high rate of morbidity-mortality worldwide. However, treating the different diseases generated by microorganisms requires a gradual increase in acquired resistance when applying or using them against various antibiotic therapies. Resistance is caused by various molecular mechanisms of microorganisms, thus reducing their effectiveness. Consequently, there is a need to search for new opportunities through natural sources with antimicrobial activity. One alternative is using peptides present in different scorpion venoms, specifically from the Buthidae family. Different peptides with biological activity in microorganisms have been characterized as preventing their growth or inhibiting their replication. Therefore, they represent an alternative to be used in the design and development of new-generation antimicrobial drugs in different types of microorganisms, such as bacteria, fungi, viruses, and parasites. Essential aspects for its disclosure, as shown in this review, are the studies carried out on different types of peptides in scorpion venoms with activity against pathogenic microorganisms, highlighting their high therapeutic potential.

Intraspecific Differences in the Venom of Crotalus durissus cumanensis from Colombia.

Biochemical and biological differences in the venom of Crotalus durissus cumanensis from three ecoregions of Colombia were evaluated. Rattlesnakes were collected from the geographic areas of Magdalena Medio (MM), Caribe (CA) and Orinoquía (OR). All three regionally distributed venoms contain proteases, PLA2s and the basic subunit of crotoxin. However, only crotamine was detected in the CA venom. The highest lethality, coagulant, phospholipase A2 and hyaluronidase activities were found in the MM venom. Also, some differences, observed by western blot and immunoaffinity, were found in all three venoms when using commercial antivenoms. Furthermore, all three eco-regional venoms showed intraspecific variability, considering the differences in the abundance and intensity of their components, in addition to the activity and response to commercial antivenoms.

Structural and functional characterization of toxic peptides purified from the venom of the Colombian scorpion Tityus macrochirus.

The soluble venom of the scorpion Tityus macrochirus was separated by chromatographic procedures and three homogeneous peptides were obtained and their primary structures were determined. They were called: Tma1-Tma3, from the abbreviated name of the scorpion. Tma1 is a peptide containing 65 amino acids with four disulfide linkages and a molecular weight of 7386.2 Da. It is a mammalian toxin, shown to affect human sodium-channels sub-types hNav1.6 and hNav1.4. Tma2 and Tma3 are peptides containing 69 amino acids linked by four disulfide bonds, molecular weights 7819.7 and 7830.0 Da, respectively. They do not affect human sodium-channels but are lethal to insects (crickets). A phylogenic analysis of the three peptides and those of other toxic peptides isolated from the genus Tityus and Centruroides were grouped together and analyzed, permitting to obtain a topology with two main clades, one includes most sodium-channel anti-insect scorpion toxins and others includes mostly sodium-channel scorpion toxins anti-mammalian. Tma1 segregates among a group of well-studied ß-class toxins of other Tityus species such as T. discrepans, T. obscurus and T. pachyurus. Tma2 and Tma3 are associated with anti-insect toxins, particularly with one of T. obscurus. This phylogenetic analysis confirms and enforces our experimental results obtained with these three new sodium-channel scorpion toxins.

Evaluación preliminar de actividad antibacteriana in vitro del veneno de escorpión Hadruroides charcasus (Karsch, 1879) contra Pseudomonas aeruginosa y Staphylococcus aureus / Preliminary evaluation of the in vitro antibacterial activity of the scorpion venom Hadruroides charcasus (Karsch, 1879) against Pseudomonas aeruginosa and Staphylococcus aureus

Objetivo: Evaluar la actividad antibacteriana in vitro veneno de Hadruroides charcasus contra a Pseudomonas aeruginosa y Staphylococcus aureus. Material y métodos. Por estimulación eléctrica se obtuvo el veneno del escorpión H. charcasus y posteriormente fue cuantificado por el método de relación de absorbancias, [mg/mL]=(1,56 x Abs 280nm) ­ (0,76 x Abs 260nm). Se realizó electroforesis, en condiciones desnaturantes (PAGE-SDS),usando un gel del 10 % y 12 % de entrecruzamiento. A través del sistema Amicon® Ultra ­ 0.5, se hizo laconcentración de las fracciones de proteínas y péptidos. Para evaluar la actividad antibacteriana se empleó cepasde P. aeruginosa y S. aureus, se hizo el método de microdilución en microplaca de 96 pozos para determinar laconcentración mínima inhibitoria (CMI). Resultados. La fracción soluble del veneno total presentó unaconcentración de 2,26 mg/mL y por PAGE-SDS, se observaron bandas con un rango peso molecular entre 7,0 ­ 9,1kDa. Se obtuvo una CMI de 0,07 mg/mL y de 0,565 mg/mL para P. aeruginosa y S. aureus una CMI de 0,035 mg/mLConclusión. el veneno del escorpión H. charcasus mostró actividad antibacteriana, con una concentración mínimainhibitoria diferente para cepas tanto S. aureus como para P. aeruginosa.

Objetive: To evaluate the in vitro antibacterial activity of Hadruroides charcasus venom against Pseudomonasaeruginosa and Staphylococcus aureus. Material andmethods. The venom of the scorpion H. charcasus wasobtained by electrical stimulation and subsequently it was quantified by the absorbance ratio method, [mg /mL] = (1.56 x Abs 280nm) - (0.76 x Abs 260nm).Electrophoresis was performed under denaturingconditions (PAGE-SDS), using a 10% gel and 12% crosslinking. Through the Amicon® Ultra-0.5 system,the concentration of protein and peptide fractions was made. To evaluate the antibacterial activity, strains of P. aeruginosa and S. aureus were used; the microdilution method was carried out in a 96-well microplate to determine the minimum inhibitory concentration (MIC). Results The soluble fraction of the total venom showed a concentration of 2,26 mg/mL and by PAGESDS, bands with a molecular weight range between 7.0- 9.1 kDa were observed. An MIC of 0,07 mg / mL and 0,565 mg / mL for P. aeruginosa and S. aureus was obtained with a MIC of 0,035 mg/mL. Conclusion. The scorpion venom H. charcasus showed antibacterial activity, with a different minimum inhibitory concentration for both S. aureus and P. aeruginosa strains.

Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

Laccases catalyze the oxidation of various aromatic organic compounds concomitantly with molecular oxygen reduction to water. Triphenylmethane dyes are synthetic compounds widely used in diverse industries. Their removal from effluents is difficult, due to their high degree of structural complexity; hence, their high concentration in effluents cause a negative impact on the environment. In the present work, molecular docking was used to evaluate interactions between rGlLCC1 or rPOXA 1B enzymes with Crystal Violet (CV) or Malachite Green (MG) dyes. In addition, removal tests of the two dyes were performed. Van der Waals interactions were obtained for only the CV dye for both GlLCC1 and POXA 1B enzymes. Nevertheless, in the GlLCC1 model, two π-π interactions were observed. For the MG dye only, Van der Waals interactions were obtained. Moreover, amino acid composition interacting in each model with each dye was similar. It is important to highlight that by molecular docking, none of the estimated ligand configurations generated hydrogen bonds. Thus, explaining the difficulty to degrade CV and MG. Regarding CV, maximum decolorization percentage was 23.6 ± 1.0% using Ganoderma lucidum supernatant and 5.0 ± 0.5% with Pleurotus ostreatus supernatant. When using recombinant laccase enzyme concentrates, decolorization percentages were 9.9 ± 0.1 and 7.5 ± 1.0% for rGlLCC1 and rPOXA 1B, respectively. On the other hand, for the MG dye, maximum decolorization percentages were 52.1 ± 5.1 and 2.3 ± 0.2% using G. lucidum and P. ostreatus concentrates, respectively. Whereas with recombinant laccase enzymatic concentrates, values of 9.4 ± 0.8% were obtained, with rGlLCC1, and 2.1 ± 0.1% when using rPOXA 1B. These findings represent an important step in bioremediation processes improvement and efficiency of industry-generated products, using environmentally friendly alternatives.

Correction to: Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

The original version of this article unfortunately contained a mistake. The replacement image of Fig. 4 provided by the first corresponding author, Aura M. Pedroza-Rodríguez, is incorrect and that the originally submitted Fig. 4 should have been retained. The original article has been corrected.

Purificación parcial de péptidos presentes en el veneno del escorpión Tityus macrochirus (Buthidae) y evaluación preliminar de su actividad citotóxica / Partial purification of peptides present in the Tityus macrochirus (Buthidae) scorpion venom and preliminary assessment of their cytotoxicity

RESUMEN Introducción. El veneno del escorpión posee péptidos con actividad neurotóxica que actúan principalmente en los canales iónicos del sistema nervioso de insectos y mamíferos. También se ha establecido su acción citolítica y anticancerígena, características biológicas que aún no se han explorado en el veneno del escorpión Tityus macrochirus. Objetivo. Evaluar si tanto el veneno total de T. macrochirus como la fracción de péptidos parcialmente purificados disminuyen el porcentaje de viabilidad de diferentes líneas celulares provenientes de tumores. Materiales y métodos. Mediante métodos cromatográficos, electroforéticos y de ultrafiltración con membranas de Amicon Ultra 0.5®, se identificaron y purificaron parcialmente los péptidos del veneno de T. macrochirus obtenido mediante estimulación eléctrica. Los ensayos de actividad citotóxica del veneno y de la fracción de péptidos se hicieron en líneas celulares provenientes de tumores con el método colorimétrico de reducción de la sal de tetrazolio (Mossman's Tetrazole Test, MTT). Resultados. El veneno de T. macrochirus posee péptidos con pesos moleculares entre 3 y 10 kDa, los cuales se purificaron parcialmente mediante ultrafiltración y se evaluaron mediante cromatografía líquida de alta resolución en fase inversa (Reverse Phase-High Pressure Liquid Chromatography, RP-HPLC). Los ensayos de citotoxicidad del veneno total de T. macrochirus evidenciaron una mayor disminución de la viabilidad en la línea celular PC3 que en las demás líneas celulares evaluadas, en tanto que la fracción parcialmente purificada de péptidos logró disminuir la viabilidad de la línea celular HeLa. Conclusión. Los péptidos del veneno de T. macrochirus presentaron actividad citotóxica en algunas de las líneas celulares provenientes de tumores, y se observó algún grado de selectividad frente a ellas.

ABSTRACT Introduction: Scorpion venom contains peptides with neurotoxic action primarily active on ion channels in the nervous system of insects and mammals. They are also characterized as cytolytic and anticancer, biological characteristics that have not yet been reported for the Tityus macrochirus venom. Objective: To assess if the total T. macrochirus venom and the fraction of partially purified peptides decrease the viability of various tumor-derived cell lines. Materials and methods: The scorpion venom was collected by electrical stimulation and, subsequently, subjected to chromatography, electrophoresis, and ultrafiltration with Amicon Ultra 0.5® membranes for the partial identification and purification of its peptides. The cytotoxic activity of the venom and the peptides fraction trials on tumor-derived cell lines were carried out by the MTT method. Results: The T. macrochirus scorpion venom has peptides with molecular weights ranging between 3 and 10 kDa. They were partially purified using the ultrafiltration technique, and assessed by the RP-HPLC method. Cytotoxicity trials with the whole T. macrochirus venom showed a higher viability decrease on the PC3 cell line compared to the other cell lines assessed, while the partially purified peptides decreased the HeLa cell line viability. Conclusion: Peptides in the T. macrochirus scorpion venom showed cytotoxic activity on some tumor-derived cell lines. We observed some degree of selectivity against other cell lines assessed.

[Partial purification of peptides present in the Tityus macrochirus (Buthidae) scorpion venom and preliminary assessment of their cytotoxicity].

INTRODUCTION: Scorpion venom contains peptides with neurotoxic action primarily active on ion channels in the nervous system of insects and mammals. They are also characterized as cytolytic and anticancer, biological characteristics that have not yet been reported for the Tityus macrochirus venom. OBJECTIVE: To assess if the total T. macrochirus venom and the fraction of partially purified peptides decrease the viability of various tumor-derived cell lines. MATERIALS AND METHODS: The scorpion venom was collected by electrical stimulation and, subsequently, subjected to chromatography, electrophoresis, and ultrafiltration with Amicon Ultra 0.5® membranes for the partial identification and purification of its peptides. The cytotoxic activity of the venom and the peptides fraction trials on tumor-derived cell lines were carried out by the MTT method. RESULTS: The T. macrochirus scorpion venom has peptides with molecular weights ranging between 3 and 10 kDa. They were partially purified using the ultrafiltration technique, and assessed by the RP-HPLC method. Cytotoxicity trials with the whole T. macrochirus venom showed a higher viability decrease on the PC3 cell line compared to the other cell lines assessed, while the partially purified peptides decreased the HeLa cell line viability. CONCLUSION: Peptides in the T. macrochirus scorpion venom showed cytotoxic activity on some tumorderived cell lines. We observed some degree of selectivity against other cell lines assessed.

Antagonistic action on NMDA/GluN2B mediated currents of two peptides that were conantokin-G structure-based designed.

BACKGROUND: The GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAr) modulates many physiological processes including learning, memory, and pain. Excessive increase in NMDAr/GluN2B activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. Thus there is an interest in identifying NMDAr antagonists that interact specifically with the GluN2B subunit. Recently based on structural analysis between the GluN2B subunit and conantokin-G, a toxin that interacts selectively with the GluN2B subunit, we designed various peptides that are predicted to act as NMDAr antagonists by interacting with the GluN2B subunit. In this study we tested this prediction for two of these peptides EAR16 and EAR18. RESULTS: The effects of EAR16 and EAR18 in NMDA-evoked currents were measured in cultured rat embryonic hippocampal neurons and in HEK-293 cells expressing recombinant NMDAr comprised of GluN1a-GluN2A or GluN1a-GluN2B subunits. In hippocampal neurons, EAR16 and EAR18 reduced the NMDA-evoked calcium currents in a dose-dependent and reversible manner with comparable IC50 (half maximal inhibitory concentration) values of 241 and 176 µM, respectively. At 500 µM, EAR16 blocked more strongly the NMDA-evoked currents mediated by the GluN1a-GluN2B (84%) than those mediated by the GluN1a-GluN2A (50%) subunits. At 500 µM, EAR18 blocked to a similar extent the NMDA-evoked currents mediated by the GluN1a-GluN2B (62%) and the GluN1a-GluN2A (55%) subunits. CONCLUSIONS: The newly designed EAR16 and EAR18 peptides were shown to block in reversible manner NMDA-evoked currents, and EAR16 showed a stronger selectivity for GluN2B than for GluN2A.

"In Silico" Characterization of 3-Phytase A and 3-Phytase B from Aspergillus niger.

Phytases are used for feeding monogastric animals, because they hydrolyze phytic acid generating inorganic phosphate. Aspergillus niger 3-phytase A (PDB: 3K4Q) and 3-phytase B (PDB: 1QFX) were characterized using bioinformatic tools. Results showed that both enzymes have highly conserved catalytic pockets, supporting their classification as histidine acid phosphatases. 2D structures consist of 43% alpha-helix, 12% beta-sheet, and 45% others and 38% alpha-helix, 12% beta-sheet, and 50% others, respectively, and pI 4.94 and 4.60, aliphatic index 72.25 and 70.26 and average hydrophobicity of -0,304 and -0.330, respectively, suggesting aqueous media interaction. Glycosylation and glycation sites allowed detecting zones that can affect folding and biological activity, suggesting fragmentation. Docking showed that H59 and H63 act as nucleophiles and that D339 and D319 are proton donor residues. MW of 3K4Q (48.84 kDa) and 1QFX (50.78 kDa) is similar; 1QFX forms homodimers which will originate homotetramers with several catalytic center accessible to the ligand. 3K4Q is less stable (instability index 45.41) than 1QFX (instability index 33.66), but the estimated lifespan for 3K4Q is superior. Van der Waals interactions generate hydrogen bonds between the active center and O2 or H of the phytic acid phosphate groups, providing greater stability to these temporal molecular interactions.

Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.

Structural features of the two-component systemLisR/LisK suggests multiple responses for the adaptation and survival of Listeria monocytogenes

Se caracterizó la estructura del sistema deregulación de dos componentes LisR/LisK de Listeriamonocytogenes. Se emplearon herramientas bioinformáticas ybases de datos para predecir la estructura e interacciones delas dos proteínas y se modelaron. Los resultados predicenque la proteína LisK está embebida en la membrana celulary su composición modular (dominios HAMP, histidinakinasa and ATPasa) está asociada a su autofosforilación(His-266). Un efecto estímulo respuesta determina lapropagación secuencial de la señal desde la membranacelular hacia componentes citoplasmáticos. A su vez, sepredice que LisR es una proteína citosólica con un dominiode receptor (homólogo a cheY) que incluye el residuofosfo-aceptor (Asp-52) y el dominio de unión a ADN,el cual puede permitir la transmisión de una respuestaespecífica a nivel transcripcional. Los componentes LisR/LisK han sido bioquímica y funcionalmente caracterizadosexperimentalmente en la patofisiología de otros bacilos. Espor ello, que la aproximación de los resultados basados enestructura-función podría facilitar el diseño de inhibidoresespecíficos...

Here, we characterized the structure of the two-component regulatory system, LisR/LisK, in Listeriamonocytogenes. To predict the structure of both proteins and the relationship between them, we employedseveral bioinformatic tools and databases. Based on our results, LisK protein is embedded in the cellmembrane and its modular composition (HAMP, histidine kinase and ATPase domains) is associatedwith its autophosphorylation (His-266). A stimulus-response likely determines the sequential signalpropagation from the bacterial cell surface to its cytoplasmic components. According to our results,LisR is a cytoplasmic protein with a receptor domain (homologous to CheY) that comprises a phosphoacceptorresidue (Asp-52) and a DNA-binding domain, which may allow the transmission of a specifictranscriptional response. LisR/LisK has been experimentally characterized both biochemically andfunctionally in other Bacilli pathophysiology; our structure-function approach may facilitate the design ofsuitable inhibitors...

O objetivo do estudo foi caracterizarestruturalmente o sistema de regulação de dois componentesLisR/LisK de Listeria monocytogenes. Foram utilizadasdiversas ferramentas de bioinformática e bancos de dadospara predizer a estrutura das duas proteínas, modelálase prever suas interações. Os resultados predizemque a proteína Lisk está incorporada na composição damembrana celular e sua composição modular (domíniosHAMP, histidina quinase e ATPase) está associada com asua autofosforilação (His-266). Um efeito de estímulo eresposta determina a propagação sequencial do sinal a partirda membrana celular em componentes citoplasmáticos. Osresultados predizem que LisR é uma proteína citosólicacom um domínio recetor (homólogo a CheY) que inclui oresíduo fosfo-aceitador (Asp-52) e o domínio de ligação aoADN, o que pode permitir a transmissão de uma respostaespecífica a nível transcricional. Como LisR/Lisk foi,química e funcionalmente, caracterizada experimentalmentena fisiopatologia de outros bacilos, esta abordagem baseadana estrutura-função pode facilitar a conceção de inibidoresespecíficos...

PRODUCTION AND PURIFICATION OF IgY ANTIBODIES AS A NOVEL TOOL TO PURIFY THE NR1 SUBUNIT OF NMDA RECEPTOR / PRODUCCIÓN Y PURIFICACIÓN DE ANTICUERPOS IgY COMO UNA HERRAMIENTA NOVEDOSA PARA PURIFICAR LA SUBUNIDAD NR1 DEL RECEPTOR NMDA / PRODUÇÂO E PURIFICAÇÂO DE ANTICORPOS IgY COMO UMA FERRAMENTA INOVADORA PARA PURIFICAR O RECEPTOR NMDA SUBUNIDADE NR1

Producing polyclonal antibodies (IgY) in chickens has advantages over those obtained in other animal models, since they have been used as a tool for studying different proteins (NMDA glutamate receptor in our case, specifically the NR1 subunit). We produced specific antibodies against expression products by the alternative splicing of the gene encoding NMDA receptor NR1 subunit in adult rat brain. Three peptides corresponding to the splicing sites (N1, C1 and C2' cassettes) were designed, synthesised and used individually as antigens in hens. Specific immunoglobulins were purified from yolks. The antibodies were then used for purifying the NMDA receptor NR1 subunit using affinity chromatography coupling the three antibodies to the support.

La producción de anticuerpos policlonales en gallinas (IgY) tiene ventajas sobre anticuerpos obtenidos en otros modelos animales y se han empleado como una nueva herramienta para estudiar diferentes proteínas (el receptor de glutamate tipo NMDA en nuestro caso, específicamente la subunidad NR1). Produjimos anticuerpos específicos contra productos de expresión por splicing alternativo del gen que codifica la subunidad NR1 del receptor tipo NMDA en el cerebro de rata adulta. Se diseñaron 3 péptidos correspondientes a los sitios de splicing del gen (conocidos como casetes N1, C1 y C2'), se sintetizaron y se usaron individualmente como antígenos en gallinas. Inmunoglobulinas específicas se purificaron de las yemas. Los anticuerpos se usaron para purificar la subunidad NR1 del receptor tipo NMDA usando cromatografía de afinidad, a través del acople de los tres anticuerpos al soporte.

A produção de anticorpos policlonais (IgY) em galinhas tem vantagens sobre os obtidos em outros modelos animais e eles têm sido usados como uma ferramenta para o estudo de proteínas diferentes (NMDA receptor de glutamato no nosso caso, especificamente a subunidade NR1). Nós produzimos anticorpos específicos contra produtos de expressão pela splicing alternativo do gene que codifica receptor NMDA subunidade NR1 no cérebro de ratos adultos. Três peptídeos correspondentes aos locais de splicing (N1, C1 e C2' cassetes) foram concebidos, sintetizados e utilizados individualmente como antígenos em galinhas. Imunoglobulinas específicas foram purificadas a partir de gemas. Os anticorpos foram então usados para purificar o receptor NMDA subunidade NR1 utilizando cromatografia de afinidade, por meio da junção dos três anticorpos ao suporte.

Estudio computacional de las relaciones evolutivas de los receptores ionotrópicos NMDA, AMPA y kainato en cuatro especies de primates / Computational study of the evolutionary relationships of the ionotropic receptors NMDA, AMPA and kainate in four species of primates / Estudo computacional das relações evolutivas dos receptores ionotrópicos NMDA, AMPA e cainato em quatro espécies de primatas

Objetivo. Identificar la influencia de los cambios respecto a la estructura secundaria y a la relación evolutiva de los receptores NMDA, AMPA Y KAINATO en las especies Homo sapiens, Pan troglodytes, Pongo pygmaeus, y Macaca mulata. Materiales y métodos. Se recopilaron 91 secuencias correspondientes a los receptores NMDA, AMPA y Kainato y se sometieron a los programas de predicción de estructura secundaria, sitios de fosforilación, alineamientos múltiples, selección del modelo de evolución y predicción filogenética. Resultados. Se encontró que las subunidades GLUR5, NR2A, NR2C y NR3A presentaron cambios de estructura en la región C-terminal y aparición o pérdida de sitios de fosforilación en esta zona. Adicionalmente la predicción filogenética nos propone que las subunidades NR2 de NMDA son las más cercanas al nodo ancestral que da origen a los demás subunidades. Conclusiones. Los cambios de estructura y sitios de fosforilación en las subunidades GLUR5, NR2A, NR2C y NR3A nos sugieren variaciones en la interacción de la región C-terminal con proteínas quinasas y con proteínas con dominios PDZ lo cual podría afectar el tráfico y anclaje de las subunidades. Por otra parte la predicción filogenética nos propone que los cambios que se presentaron en las subunidades NR2 dieron origen a las demás subunidades de los receptores ionotrópicos de glutamato, básicamente porque son las subunidades de NMDA y en particular NR2D las que se encuentran más estrechamente relacionadas con el nodo ancestral que posiblemente dio origen a los iGluRs.

Objective. To identify the influence of changes on the secondary structure and evolutionary relationship of NMDA, AMPA and kainate receptors in Homo sapiens, Pan troglodytes, Pongo pygmaeus and Macaca mulatta. Materials and methods. We identified 91 sequences for NMDA, AMPA and kainate receptors and analyzed with software for predicting secondary structure, phosphorylation sites, multiple alignments, selection of protein evolution models and phylogenetic prediction. Results. We found that subunits GLUR5, NR2A, NR2C and NR3A showed structural changes in the C-terminal region and formation or loss of phosphorylation sites in this zone. Additionally the phylogenetic prediction suggests that the NMDA NR2 subunits are the closest to the ancestral node that gives rise to the other subunits. Conclusions. Changes in structure and phosphorylation sites in GLUR5, NR2A, NR2C and NR3A subunits suggest variations in the interaction of the C-terminal region with kinase proteins and with proteins with PDZ domains, which could affect the trafficking and anchoring of the subunits. On the other hand, the phylogenetic prediction suggests that the changes that occurred in the NR2 subunits gave rise to the other subunits of glutamate ionotropic receptors, primarily because the NMDA and particularly the NR2D subunits are the most closely related to the ancestral node that possibly gave rise to the iGluRs.

Objetivo. Identificar a influência das mudanças da estrutura secundária e da relação evolutiva dos receptores NMDA, AMPA e cainato nas espécies Homo sapiens, Pan troglodytes, Pongo pygmaeus e Macaca mulatta. Materiais e métodos. Foram recopiladas 91 seqüências correspondentes aos receptores NMDA, AMPA e cainato e foram submetidos a programas de predição de estrutura secundária, sítios de fosforilação, alinhamentos múltiplos, seleção do modelo de evolução e predição da filogenia. Resultados. Descobrimos que as subunidades GLUR5, NR2A, NR2C e NR3A apresentaram alterações estruturais na região C-terminal e aparecimento ou perda de sítios de fosforilação nesta área. Além disso, a predição filogenética sugere ainda que as subunidades NR2 de NMDA são as mais próximas ao nó ancestral que dá origem às demais subunidades. Conclusões. As mudanças na estrutura e nos sítios de fosforilação nas subunidades GLUR5, NR2A, NR2C e NR3A sugerem variações na interação da região C-terminal com proteínas quinase e com proteínas de domínios PDZ que poderia afetar o tráfego e fixação das subunidades. Além disso, a predição filogenética sugere que as mudanças ocorridas nas subunidades NR2 deram origem às outras subunidades de receptores ionotrópicos de glutamato, principalmente porque são subunidade NMDA e particularmente NR2D aquelas que são mais estreitamente relacionadas com o nó ancestral que provavelmente deu origem aos iGluRs.

Aproximación in silico a la evolución de la familia de genes que conforman el receptor ionotrópico de glutamato en cuatro especies de primates / In silico approach to the evolution of ionotropic glutamate receptor gene family in four primate species / Aproximação In silico á evolução da família de genes que compõem o receptor ionotrópico de Glutamato em quatro espécies de primatas

El hombre como especie posee un cerebro único en capacidades de análisis por su estructura y patrones de organización que presumiblemente son la base de la inteligencia y la habilidad de manipular el entorno. Adicionalmente, el desarrollo y la evolución del cerebro responden los procesos genéticos subyacentes. Objetivo. Presentar una aproximación al proceso evolutivo de los iGluR con el método filogenético de máxima verosimilitud (ML) y bayesiano (By). Materiales y métodos. Para lo cual se emplean métodos in silico que permiten plantear un modelo de evolución molecular y el reconocimiento cualitativo de los bloques de sintenia, para estos genes en las especies de primates (chimpancé, orangután, mono rhesus y hombre). Resultados. El Glutamato es el principal neurotransmisor y juega un papel importante en la plasticidad neuronal y la neurotoxicidad. La neurotransmisión vía glutamato es mediada por los receptores ionotrópicos de Glutamato (iGluR) tipo NMDA y no-NMDA (AMPA y KA). Se tiene que por cada inferencia filogenética obtenida, se confirma que los iGluR de los mamíferos podrían haber evolucionado a partir de un mecanismo más primitivo de señalización, por lo cual se presentan agrupaciones similares entre algunas especies de primates con roedores. Conclusión: Las secuencias de NR-2 han permanecido en una selección purificadora, y que la escala de divergencia neutral es más rápida en los primates que en los roedores; sin embargo es necesario aplicar otros estudios para confirman estas teorías de evolución.

Man as a species has a brain unique in analysis capabilities due to its structure and organizational patterns that are presumably the basis of intelligence and the ability to manipulate the environment. Additionally, the development and evolution of the brain respond underlying genetic processes. Objective. To present an approach to the evolutionary process of the iGluR with the maximum likelihood (ML) and Bayesian (By) phylogenetic analysis methods. Materials and methods. we used in silico methods to propose a model of molecular evolution and to do a qualitative recognition of synteny blocks for these genes in different species of primates (chimpanzee, orangutan, rhesus monkey and man). Results. Glutamate is the main neurotransmitter and plays an important role in neuronal plasticity and neurotoxicity. Neurotransmission via glutamate is mediated by ionotropic glutamate receptors (iGluR) NMDA type and non-NMDA type (AMPA and KA). For every phylogenetic inference, we confirmed that the iGluRs of mammals could have evolved from a primitive signaling mechanism, thus explaining similar clusters between some species of primates and rodents. Conclusion. The NR-2 sequences have been exposed to a purifying selection, and the neutral level of divergence is faster in primates than in rodents, however further studies are needed to confirm these theories of evolution.

O homem como espécie tem um cérebro único em capacidades de análise por sua estrutura e padrões de organização que, presumivelmente, são a base da inteligência e da capacidade de manipular o meio ambiente. Além ao desenvolvimento e a evolução do cérebro, respondem os processos genéticos subjacentes. Objetivo. Apresentar uma aproximação ao processo evolutivo dos iGluR com o método filogenético de máxima verossimilhança (ML) e bayesiano (By.) Materiais e métodos. Foram empregados métodos in silico que permitem apresentar um modelo de evolução molecular e o reconhecimento qualitativo dos blocos de sintenia, para estes genes das espécies de primatas (chimpanzé, orangotango, o macaco rhesus e o homem). Resultados. O Glutamato é o principal neurotransmissor e desempenha um importante papel na plasticidade neuronal e na neurotoxicidade. A neurotransmissão via Glutamato é mediada pelos receptores ionotrópicos de Glutamato (iGluR) do tipo NMDA e no-NMDA (AMPA e KA). Foi observado que por cada inferência filogenética obtida, confirmase que os iGluR dos mamíferos poderiam ter evoluído a partir de um mecanismo mais primitivo de sinalização, pelo qual se apresentam agrupações semelhantes entre algumas espécies de primatas com roedores. Conclusão. As seqüências de NR-2 têm permanecido numa seleção purificadora, e que a escala de divergência neutral é mais rápida em primatas do que em roedores; embora, é necessario realizar outras pesquisas para confirmar estas teorias da evolução.

Immunolocalization and biochemical characterization of N-methyl-D-aspartate receptor subunit NR1 from rat brain.

The N-methyl-D-aspartate (NMDA) receptor subunit NR1 gene can produce eight isoforms in rat brain. A novel methodology for purifying NMDA receptor NR1 subunit from rat brain is reported here using chicken polyclonal antibodies (IgYs) against synthetic peptides corresponding to N1, C1 and C2' cassettes. The isolated protein was recognized by produced IgYs and commercial anti-NR1 IgGs, shown by MALDI-TOF MS a MW = 131,192 Da (glycosylated form); the enzymatically deglycosylated protein revealed a MW = 102,754 Da. The NMDA receptor NR1 subunit was characterized as being a heavily N-glycosylated protein. The isoelectric point was determined (6.3) as being different from that predicted for any of the isoforms (7.9-9.02). Attempts to separate the isoforms from the purified NR1 were unsuccessful, indicating the presence of just one isoform (NR1(111)). Immunohistochemistry on hippocampus regions CA1, CA3 and Dentate gyrus with anti-N1, anti-N2 and anti-C2' IgYs showed different staining intensity, depending upon the antibody assayed.