Inhibitory Effect of Gallic Acid and Its Esters on 2,2 '-Azobis(2-amidinopropane)hydrochloride (AAPH)-Induced Hemolysis and Depletion of Intracellular Glutathione in Erythrocytes

The protective effect of gallic acid and its esters, methyl, propyl, and lauryl gallate, against 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione (GSH) in erythrocytes was studied. The inhibition of hemolysis was dose-dependent, and the esters were significantly more effective than gallic acid. Gallic acid and its esters were compared with regard to their reactivity to free radicals, using the DPPH and AAPH/pyranine free-cell assays, and no significant difference was obtained. Gallic acid and its esters not only failed to inhibit the depletion of intracellular GSH in erythrocytes induced by AAPH but exacerbated it. Similarly, the oxidation of GSH by AAPH or horseradish peroxidase/H(2)O(2) in cell-free systems was exacerbated by gallic acid or gallates. This property could be involved in the recent findings on proapoptotic and pro-oxidant activities of gallates in tumor cells. We provide evidence that lipophilicity and not only radical scavenger potency is an important factor regarding the efficiency of antihemolytic substances.

Anti-Helicobacter pylori activity and immunostimulatory effect of extracts from Byrsonima crassa Nied. (Malpighiaceae)

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

Investigation of the interaction between Tc85-11 protein and antibody anti-T cruzi by AFM and amperometric measurements

This present work reports on development of an amperometric immunosensor for the diagnosis of Chagas' disease using a specific glycoprotein of the trypomastigote surface, which belongs to the Tc85-11 protein family of Trypanosoma cruzi (T cruzi). An atomically flat gold surface on a silicon substrate and gold screen-printed electrodes were functionalized with cystatrine and later activated with glutaraldehyde (GA), which was used to form covalent bonds with the purified recombinant antigen (Tc85-11). The antigen reacts with the antibody from the serum, and the affinity reaction was monitored directly using atomic force microscopy or amperometry through a secondary antibody tagged to peroxidase (HRP). Surface imaging allowed to us to differentiate the modification steps and antigen-antibody interaction allowed to distinguish the affinity reactions. In the amperometric immunosensor, peroxidase catalyses the L-2 formation in the presence of hydrogen peroxide and potassium iodide, and the reduction current intensity was measured at a given potential with screen-printed electrodes. The immunosensor was applied to sera of chagasic patients and patients having different systemic diseases. (c) 2006 Elsevier Ltd. All rights reserved.

The reactivity of oytho-methoxy-substituted catechol radicals with sulfhydryl groups: Contribution for the comprehension of the mechanism of inhibition of NADPH oxidase by apocynin

Redox processes are involved in the mechanism of action of NADPH oxidase inhibitors such as diphenyleneiodonium and apocynin. Here, we studied the structure-activity relationship for apocynin and analogous ortho-methoxy-substituted catechols as inhibitors of the NADPH oxidase in neutrophils and their reactivity with peroxidase. Aiming to alter the reduction potential, the ortho-methoxy-catechol moiety was kept constant and the substituents at para position related to the hydroxyl group were varied. Two series of compounds were employed: methoxy-catechols bearing electron-withdrawing groups (MC-W) such as apocynin, vanillin, 4-nitroguaiacol, 4-cyanoguaiacol, and methoxy-catechol bearing electron-donating groups (MC-D) such as 4-methylguaiacol and 4-ethylguaiacol. We found that MC-D were weaker inhibitors compared to MD-W. Furthermore, the radicals generated by oxidation of MC-W via MPO/H(2)O(2), but not for MC-D, were able to oxidize glutathione (GSH) as verified by the formation of thiyl radicals, depletion of GSH, and recycling of the ortho-methoxy-catechols during their oxidations. The capacity of oxidizing sulfhydryl (SH) groups was also verified when ovalbumin was incubated with MC-W, but not for MC-D. Since the effect of apocynin has been correlated with inactivation of the cytosolic fractions of the NADPH oxidase complex and its oxidation during the inhibitory process develops a special role in this process, we suggest that the close relationship between the reactivity of the radicals of MC-W compounds with thiol groups and their efficacy as NADPH oxidase inhibitor could be the chemical pathway behind the mechanism of action of apocynin and should be taken into account in the design of new and specific NADPH oxidase inhibitors. (c) 2007 Elsevier B.V. All rights reserved.

p-iodophenol-enhanced luminol chemiluminescent assay applied to discrimination between acute lymphoblastic and minimally differentiated acute myeloid (FAB-M0) or acute megakaryoblastic (FAB-M7) leukemias

Introduction: In this report, we propose the application of the p-iodophenol-enhanced luminol chemiluminescent technique to the determination of peroxidase (myeloperoxidase and/or platelet peroxidase) activity in blasts of minimally differentiated acute myeloblastic leukemia (AML-M0) and acute megakaryoblastic leukemia (AML-M7).Methods: the frozen blast cells from 29 patients were thawed and submitted to the optimized protocol.Results: All cases of AML-M7 and AML-M0 exhibited integrated light emission greater than 73 (10(2) mV x s), which was the arbitrary cutoff point set for the discrimination between AML and acute lymphoblastic leukemia (ALL) (mean + 3 x s.d. of ALL samples, n = 10). In addition, five out of seven cases of AML-M0 showed results above the Cutoff point.Conclusion: This highly sensitive enhanced chemiluminescent technique may be applied to discriminate between ALL and AML-M7 or AML-M1 cases, and most AML-M0 cases. It is very simple, cheap and easy to perform compared to other procedures used to measure MPO activity in AML-leukemias including AML-M7 and AML-M0.

Selective activity of butyrylcholinesterase in serum by a chemiluminescent assay

In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H2O2 system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-scruin-HRP-H2O2 System consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at less than or equal to4 degreesC. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O-2 uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta -methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyryleholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme. Copyright (C) 2001 John Wiley & Sons, Ltd.

Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45 V versus Ag/AgCl using H2O2 and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease. (c) 2006 Elsevier B.V. All rights reserved.

Immunosensor for the diagnosis of Chagas' disease

Trypanosoma cruzi proteins from epimastigote membranes, herein referred as antigens, have been used for the construction of an amperometric immunosensor for serological diagnosis of Chagas' disease. The proteins used had a molecular mass ranging from 30 to 100 kDa. The gold electrode was treated with cysteamine and glutaraldehyde prior to antigen immobilization. Antibodies present in the serum of patients with Chagas' disease were captured by the immobilized antigens and the affinity interaction was monitored by chronoamperometry at a potential of -400 mV (versus Ag pseudo-reference electrode) using peroxidase-labeled IgG conjugate and hydrogen peroxide, iodide substrate. The incubation time to allow maximum antigen-antibody and antibody-peroxidase-labeled IgG interactions was 20 min with a reactivity threshold at -0.104 mu A. (c) 2004 Elsevier B.V. All rights reserved.

Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction

Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 ± 2.46 and 21.22 ± 2.44 µg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 µg/ml) and 24 h (0.5, 1, and 5 µg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 µg/ml lipopolysaccharide (LPS) solution varied from 14.47 ± 1.46 to 22.35 ± 1.94 µmol/l and 13.50 ± 1.42 to 20.44 ± 1.40 µmol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.

Otimização de ELISA empregando a proteína Tc85-11 e planejamento fatorial

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

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin

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

Optimized synthesis of the melatonin metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência espaço-temporal e fisiológica na absorção de nutrientes e elementos tóxicos por Eichhornia crassipes visando o uso adequado da sua biomassa: o caso do Rio Apodi/Mossoró - RN

This study aims to determine the amount of nutrients and toxic elements in aquatic macrophytes of species Eichhornia crassipes present in River Apodi/Mossoró - RN and check some of the possibilities of using the biomass produced, based on the influence of space - temporal and physiological absorption of nutrients by plants. For this, was determined: Leaf area, Leaf wet mass, Leaf dry mass, Real humidity, Apparent humidity, Ash, Total nitrogen, Crude protein, Calcium, Magnesium, Potassium, Total phosphorus, Sodium, Iron, Copper, Manganese, Zinc, Nickel, Cobalt, Aluminum, Cadmium, Lead and Total chromium at different times, 2 sampling points and 2 parts of plants (leaves and roots). The results show that the levels of nutrients, protein and toxic elements present in plant tissue of Eichhornia crassipes are influenced by spatial, temporal and physiological variability. In general, because the maximum values in the dry matter for total nitrogen (4.4088 g/100g), crude protein (27.5549 g/100g), total phosphorus (0.642 g/100 g), calcium (1.444 g/100g), magnesium (0.732 g/100 g), potassium (7.51 g/100 g), copper (4.4279 mg/100g), manganese (322.668 mg/100g), sodium (1.39 g/100g), iron (194.169 mg/100g) and zinc (3.5836 mg/100g), there was the possibility of using biomass of Eichhornia crassipes for various purposes such as in food animal, products production for human consumption, organic fertilizers, fabrication of brick low cost, and crafts. For all these applications requires a control of the levels of substances in plant tissue. Based on the levels of nutrients and crude protein, the younger plants (0 Month) would be best to have their biomass used. Moreover, one factor that contributes to the use of larger plants (6 Months), the levels of toxic elements which have significantly small or below the detection limit. Therefore, further studies quantifying the biomass produced/m2 at 0 and 6 months are needed for a more correct choice for the best time of harvest

Análise bromatológica de carnes antes e após o processamento para obtenção de charque

The process of salting and drying in the sun is used to preserve meat since the beginning of civilization. There is evidence that this preservation technique has arisen in Egypt, between 4,000 and 5,000 years ago. In our country, according to literature, was the first industrial product that gave the appearance of beef jerky, beef being produced, where about 70% to 75% of the muscle is composed of water, where it will be around 45% as a final product, according to the law in his article RIISPOA No. 432 provides that the jerky should contain no more than this amount of moisture in the muscular portion, or more than 15% of total ash with tolerance of up to 5% variation . Besides this parameter, proteins, lipids, ash, and minerals were analyzed in samples before and after the manufacturing process to know the content of these nutrients. Since these are considered important in product quality, thus the concentration in these samples, respectively, in the flesh Front (CD and CHD) before and after the manufacturing process for humidity were respectively 75.28% and 47.38% , the protein was 14.17 and 22.20 g / 100 g sample, 6.360 and 4.251 of lipids g/100g of the sample, and the ashes 0.974 9.144 g/100g sample, minerals like calcium and 4.074 30 , 06 ppm, sódio0, 055 and 5.401 g / L, sodium chloride, 0.139 and 13.74 g / L, potassium 237.5 and 166.8 ppm, 1.721 and 3.295 ppm iron, 0.143 and 0.135 ppm phosphorus, zinc and 4.690 6.905 ppm; magnésio14, 63 e13, 75 ppm manganese .017 e0, 007ppm, copper 0.057 and 0.039 ppm in the case of needle-type meat (CPA and CHPA), 68.04% and 44.17%, protein 13 , 72, and 24.42 g/100g of sample, 1.137 in the ash and 12.68 g / 100g of sample, and the minerals calcium 17.11 and 12.89 ppm; sódio0, 123 and 4.871 g / L, sodium chloride 0.312 and 12.39 g / L, potassium 305.3 and 182.1 ppm; ferro1, 817 and 1.513 ppm, 0.273 and 0.139 ppm phosphorus, zinc 6.305 and 4.783 ppm, 27.95 and 15.85 ppm magnesium, manganese and 0.025 0.011 ppm, 0.057 and 0.143 ppm copper and chromium 0.014 and 0.068 ppm

Estudo das características magnéticas e absorvedoras das ferritas de nizn, niznmn, mnzn, nimg, nicuzn e nicuznmg obtidas via método do citrato precursor

Were synthesized systems Ni0,5Zn0,5Fe2O4, i0,2Zn0,5Mn0,3Fe2O4, Mn0,5Zn0,5Fe2O4, Ni0,5Mg0,5Fe2O4, Ni0,2Cu0,3Zn0,5Fe2O4 and Ni0,2Cu0,3Zn0,5Mg0,08Fe2O4, the precursors citrate method. The decomposition of the precursors was studied by thermogravimetric analysis and spectroscopy in the infrared region, the temperature of 350°C/3h. The evolution of the phases formed after calcinations at 350, 500, 900 and 1100ºC/3h was accompanied by X-ray diffraction using the Rietveld refinement to better identify the structures formed. The materials were also analyzed by scanning electron microscopy, magnetic measurements and analysis of the reflectivity of the material. The samples calcined at different temperatures showed an increase of crystallinity with increasing calcination temperature, verifying that for some compositions at temperatures above 500°C precipitates of second phase such as hematite and CuO. The compositions of manganese present in the structure diffusion processes slower due to the ionic radius of manganese is greater than for other ions substitutes, a fact that delays the stabilization of spinel structure and promotes the precipitation of second phase. The compositions presented with copper precipitation CuO phase at a temperature of 900 and 1100ºC/3h This occurs according to the literature because the concentration of copper in the structure is greater than 0.25 mol%. The magnetic measurements revealed features of a soft ferrimagnetic material, resulting in better magnetic properties for the NiZn ferrite and NiCuZnMg at high temperatures. The reflectivity measurements showed greater absorption of electromagnetic radiation in the microwave band for the samples calcined at 1100ºC/3h, which has higher crystallite size and consequently the formation of multi-domain, increasing the magnetization of the material. The results of absorption agreed with the magnetic measurements, indicating among the ferrites studied, those of NiZn and NiCuZnMg as better absorbing the incident radiation.