Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

BIOSSENSOR ELETROQUÍMICO BASEADO NA ENZIMA TIROSINASE PARA A DETERMINAÇÃO DE FENOL EM EFLUENTES

AbstractThis work describes the development of a biosensor based on the tyrosinase enzyme (Tyr) for the determination of phenol (PHEN) in laboratory effluent samples derived from ammoniacal nitrogen analysis of the water samples from the Muquém dam in the city of Cariús, CE, using square-wave voltammetry (SWV). The electrode modification consisted of the immobilization of gold nanoparticles, multi-walled carbon nanotubes, cobalt phthalocyanine, and Tyr on a glassy carbon electrode. The electrolyte, pH, enzyme quantity, and voltammetric parameters were optimized to detect PHEN. The analytical curves presented a linear range from 4.97 × 10-6 mol L-1 to 6.10 × 10-5 mol L-1, and the detection limit (DL) and quantitation limit (QL) values were 4.81 × 10-6 mol L-1 and 4.97 × 10-6mol L-1, respectively. The repetition of measurements with the same biosensor and repetition for three other prepared biosensors exhibited a relative standard deviation (RSD) of 5.50 and 1.75%, respectively. The percentage recovery of PHEN in effluent samples varied from 86.40 to 105.04%. The stability of the biosensor was evaluated (at 21 days) with satisfactory results, showing 97.86% of the initial response. Moreover, the DL and recovery percentages agreed with the established values from CONAMA and ABNT, respectively. Thus, the electrode configuration developed seems a promising tool in the detection and quantification of PHEN in complex samples.

Concentrações plasmáticas de triptamina, tiramina e feniletilamina em eqüinos sob efeitos de sobrecarga de carboidratos e antiinflamatórios não esteroidais

As concentrações plasmáticas das aminas triptamina (TRP), tyramina (TYR) e pheniletilamina (PEA) foram determinadas por cromatografia gasosa (CG) de 20 eqüinos sob efeito de sobrecarga por carboidratos (SC). Após 36h da SC os animais foram aleatoriamente divididos em quatro grupos (n=5) e receberam a cada 12h por via iv: solução salina 10mL (GC), ketoprofeno 2,2mg/kg (GK), fenilbutazona 4,4mg/kg (GF) e flunixin meglumine 1,1mg/kg (GFM). As concentrações das aminas TYR e PEA variaram de 0,18 a 164,2mg/L, com diferenças nos tempos avaliados, mas não entre os tratamentos (p<0,01). A concentração plasmática de TRP apresentou diferenças entre os tempos e também entre os tratamentos. O GC diferiu dos demais nos momentos 48h e 60h e as concentrações nos grupos GK e GFM foram menores que nos grupos GF e GC às 72h (P= 0,0012). Conclui-se que nas doses utilizadas os antiinflamatórios não esteroidais avaliados não interferem nas concentrações de TYR e PEA. Entretanto, o ketoprofeno e o flunixin meglumine foram efetivos em diminuir a concentração plasmática de TRP.

Simulação da soja geneticamente modificada tolerante ao glyphosate por meio do cultivo de explantes

O objetivo do experimento consistiu na simulação in vitro da soja transgênica tolerante ao glyphosate, através do cultivo de explantes em meios de cultura contendo aminoácidos aromáticos. As avaliações basearam-se nos efeitos do glyphosate sobre sementes oriundas de explantes de soja (Glycine max (L.) Merr.) cv. UFV-16. Para tanto, explantes de soja foram cultivados em meios de cultura líquidos com pH em torno de 5,0. Cada explante constou de um legume completamente expandido contendo duas sementes de aproximadamente 100 mg, conectada a um segmento de caule de 45 mm de comprimento. Os tratamentos testados foram: A = glutamina (Gln); B = Gln + fenilalanina (Phe) + tirosina (Tyr) + triptofano (Trp); C = Gln + glyphosate; D = Gln + Phe + Tyr + Trp + glyphosate. O experimento foi conduzido sob irradiância de 80 mmol-2s-1 a 25oC por 204 horas. Nos tratamentos que receberam aminoácidos aromáticos e glutamina, o herbicida não afetou as massas fresca e seca das sementes, como também, não afetou seus constituintes bioquímicos (proteínas, óleo, ácidos graxos, carboidratos e clorofilas). Portanto, a suplementação exógena de aminoácidos aromáticos suprime os efeitos fitotóxicos do glyphosate sobre explantes de soja, permitindo estudos sobre o seu modo de ação e metabolismo nas sementes, uma vez que os explantes se comportaram analogamente à soja transgênica não suscetível ao herbicida.

Isolation and characterization of a reserve protein from the seeds of Opuntia ficus-indica (Cactaceae)

We describe here the isolation and characterization of a major albumin from the seeds of Opuntia ficus-indica (Cactaceae). This protein has a molecular mass of 6.5 kDa and was isolated by a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of this protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The N-terminal amino acid sequence of this protein is Asp-Pro-Tyr-Trp-Glu-Gln-Arg.

Role of angiotensin II and vasopressin receptors within the supraoptic nucleus in water and sodium intake induced by the injection of angiotensin II into the medial septal area

In this study we investigated the effects of the injection into the supraoptic nucleus (SON) of non-peptide AT1- and AT2-angiotensin II (ANG II) receptor antagonists, DuP753 and PD123319, as well as of the arginine-vasopressin (AVP) receptor antagonist d(CH2)5-Tyr(Me)-AVP, on water and 3% NaCl intake induced by the injection of ANG II into the medial septal area (MSA). The effects on water or 3% NaCl intake were assessed in 30-h water-deprived or in 20-h water-deprived furosemide-treated adult male rats, respectively. The drugs were injected in 0.5 µl over 30-60 s. Controls were injected with a similar volume of 0.15 M NaCl. Antagonists were injected at doses of 20, 80 and 180 nmol. Water and sodium intake was measured over a 2-h period. Previous administration of the AT1 receptor antagonist DuP753 into the SON decreased water (65%, N = 10, P<0.01) and sodium intake (81%, N = 8, P<0.01) induced by the injection of ANG II (10 nmol) into the MSA. Neither of these responses was significantly changed by injection of the AT2-receptor antagonist PD123319 into the SON. On the other hand, while there was a decrease in water intake (45%, N = 9, P<0.01), ANG II-induced sodium intake was significantly increased (70%, N = 8, P<0.01) following injection of the V1-type vasopressin antagonist d(CH2)5-Tyr(Me)-AVP into the SON. These results suggest that both AT1 and V1 receptors within the SON may be involved in water and sodium intake induced by the activation of ANG II receptors within the MSA. Furthermore, they do not support the involvement of MSA AT2 receptors in the mediation of these responses.

Cardiovascular effects of the intracerebroventricular injection of adrenomedullin: roles of the peripheral vasopressin and central cholinergic systems

Our objective was to investigate in conscious Sprague-Dawley (6-8 weeks, 250-300 g) female rats (N = 7 in each group) the effects of intracerebroventricularly (icv) injected adrenomedullin (ADM) on blood pressure and heart rate (HR), and to determine if ADM and calcitonin gene-related peptide (CGRP) receptors, peripheral V1 receptors or the central cholinergic system play roles in these cardiovascular effects. Blood pressure and HR were observed before and for 30 min following drug injections. The following results were obtained: 1) icv ADM (750 ng/10 µL) caused an increase in both blood pressure and HR (DMAP = 11.8 ± 2.3 mmHg and ΔHR = 39.7 ± 4.8 bpm). 2) Pretreatment with a CGRP receptor antagonist (CGRP8-37) and ADM receptor antagonist (ADM22-52) blocked the effect of central ADM on blood pressure and HR. 3) The nicotinic receptor antagonist mecamylamine (25 µg/10 µL, icv) and the muscarinic receptor antagonist atropine (5 µg/10 µL, icv) prevented the stimulating effect of ADM on blood pressure. The effect of ADM on HR was blocked only by atropine (5 µg/10 µL, icv). 4) The V1 receptor antagonist [β-mercapto-β-β-cyclopentamethylenepropionyl¹, O-me-Tyr��,Arg8]-vasopressin (V2255; 10 µg/kg), that was applied intravenously, prevented the effect of ADM on blood pressure and HR. This is the first study reporting the role of specific ADM and CGRP receptors, especially the role of nicotinic and muscarinic central cholinergic receptors and the role of peripheral V1 receptors in the increasing effects of icv ADM on blood pressure and HR.