Antonio Augusto Fidalgo-Neto

In vitro inhibition of liver monooxygenases by β-ionone, 1,8-cineole, (-)-menthol and terpineol

The present study was undertaken to investigate the inhibitory effects of beta-ionone, (-)-menthol, 1,8-cineole and alpha-terpineol on liver microsomal enzymes involved in the biotransformation of xenobiotic substances. The effects of beta-ionone and the foregoing monoterpenoid compounds on the activity of pentoxyresorufin-O-depentilase (PROD), a selective marker for CYP2B1, were determined in a pool of liver microsomes prepared from phenobarbital-treated rats. On the other hand, the inhibitory effects of these substances on the activities of ethoxyresorufin-O-deethylase (EROD), a marker for CYP1A1, and methoxyresorufin-O-demethylase (MROD), a marker for CYP1A2, were investigated in a pool of hepatic microsomes from beta-naphthoflavone-treated rats. Beta-ionone caused a concentration-related reduction of PROD activity with an IC50 value as low as 0.03 microM. The analysis of alterations produced by beta-ionone on PROD kinetic parameters (Lineweaver-Burk double-reciprocal plot) suggested that inhibition is non-competitive (Ki = 89.9 nM). Although being less potent than beta-ionone, 1,8-cineole (IC50 = 4.7 microM), (-)-menthol (IC50 = 10.6 microM) and terpineol (IC50 = 14.8 microM) also proved to be in vitro inhibitors of PROD reaction. Results also revealed that beta-ionone was a weak inhibitor of EROD (IC50 >100 microM) and MROD (IC50 >200 microM). Neither 1,8-cineole nor terpineol--tested in concentrations up to 150 microM--caused any decrease of EROD activity while (-)-menthol, at a concentration as high as 160 microM, produced only a slight reduction of the reaction rate. Terpineol (up to 150 microM) did not induce any reduction of MROD activity while 1,8-cineole (IC50 >300 microM) and (-)-menthol (IC50 >300 microM) caused only slight decreases of the reaction rate. The potent inhibitory effects on CYP2B1 suggest that beta-ionone, and the other monoterpenoids tested, may interfere with the metabolism of xenobiotics which are substrates for this isoenzyme.

Activity of liver microsomal enzymes during the chronic phase of murine schistosomiasis

The effects of schistosomiasis on microsomal enzymes were studied on post-infection day 90 when accumulated damage and fibrosis are most intense but granulomatous reaction around the eggs harbored in the liver is smaller than during the earlier phases. Swiss Webster (SW) and DBA/2 mice of either sex (N = 12 per sex per group) were infected with 100 Schistosoma mansoni cercariae on postnatal day 10 and killed on post-infection day 90. Cytochrome P-450 (CYP) concentration and alkoxyresorufin-O-dealkylases (EROD, MROD, BROD, and PROD), p-nitrophenol-hydroxylase (PNPH), coumarin-7-hydroxylase (COH), and UDP-glucuronosyltransferase (UGT) activities were measured in hepatic microsomes. Age-matched mice of the same sex and strain were used as controls. In S. mansoni-infected mice, CYP1A- and 2B-mediated activities (control = 100%) were reduced in SW (EROD: male (M) 36%, female (F) 38%; MROD: M 38%, F 39%; BROD: M 46%, F 19%; PROD: M 50%, F 28%) and DBA/2 mice (EROD: M 64%, F 58%; MROD: M 60%; BROD: F 49%; PROD: M 73%) while PNPH (CYP2E1) was decreased in SW (M 31%, F 38%) but not in DBA/2 mice. COH did not differ between infected and control DBA/2 and UGT, a phase-2 enzyme, was not altered by infection. In conclusion, chronic S. mansoni infection reduced total CYP content and all CYP-mediated activities evaluated in SW mice, including those catalyzed by CYP2E1 (PNPH), CYP1A (EROD, MROD) and 2B (BROD, PROD). In DBA/2 mice, however, CYP2A5- and 2E1-mediated activities remained unchanged while total CYP content and activities mediated by other CYP isoforms were depressed during chronic schistosomiasis.

Structural and Molecular Modeling Features of P2X Receptors

Currently, adenosine 5′-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors.

Effect of Rheedia longifolia Leaf Extract and Fractions on the P2X7 Receptor In Vitro: Novel Antagonists?

Recently, the P2X(7) receptor has been reported to be associated with chronic inflammatory and neuropathic pain. Because Rheedia longifolia extract has analgesic and anti-inflammatory activity, we evaluated the in vitro inhibitory potential of methanol extract and fractions from its leaves on the P2X(7) purinergic receptor. The activity of P2X(7) was studied with a dye uptake assay and with the whole-cell patch clamp technique in mouse peritoneal macrophages treated with methanol extract of R. longifolia leaves and fractions. The dye uptake was evaluated by flow cytometry and fluorescence microscopy. The R. longifolia extract and some fractions showed an inhibitory effect on the P2X(7) purinergic receptor in a dose-dependent manner. The ethyl acetate fraction exhibited the most potent inhibitory effects. The methanol extract and the butanol fraction showed the same inhibitory effects, despite their lower potency compared with the other fractions. The R. longifolia extract and some of its fractions may be anti-inflammatory because of their inhibitory effect on the P2X(7) receptor. Further investigation is needed to determine the pattern of inhibition and selectivity. Chromatographic analysis indicated the presence of bisflavonoids in the methanol extract fractions. A member of this chemical family is the most probable active compound responsible for the P2X(7) inhibitory effects present in the R. Longifolia extract and fractions.

PHARMAVIRTUA: Educational Software for Teaching and Learning Basic Pharmacology.

information and communication technologies have become important tools for teaching scientific subjects such as anatomy and histology as well as other, nondescriptive subjects like physiology and pharmacology ([8][1]–[10][2]). Software has been used to facilitate the learning of specific concepts

Virtual immunology: Software for teaching basic immunology

As immunology continues to evolve, many educational methods have found difficulty in conveying the degree of complexity inherent in its basic principles. Today, the teaching–learning process in such areas has been improved with tools such as educational software. This article introduces “Virtual Immunology,” a software program available free of charge in Portuguese and English, which can be used by teachers and students in physiology, immunology, and cellular biology classes. We discuss the development of the initial two modules: “Organs and Lymphoid Tissues” and “Inflammation” and the use of interactive activities to provide microscopic and macroscopic understanding in immunology. Students, both graduate and undergraduate, were questioned along with university level professors about the quality of the software and intuitiveness of use, facility of navigation, and aesthetic organization using a Likert scale. An overwhelmingly satisfactory result was obtained with both students and immunology teachers. Programs such as “Virtual Immunology” are offering more interactive, multimedia approaches to complex scientific principles that increase student motivation, interest, and comprehension.

An Improved Method for P2X7R Antagonist Screening

ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimers disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC50 value of the known agonist ATP and the ICs50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 mM and 173-285 μM for ATP, BBG and OATP, respectively). The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist

Effects of pregnancy and protein-energy malnutrition on monooxygenase O-dealkylation activity in rat liver microsomes

Xenobiotic metabolism is influenced by a variety of physiological and environmental factors including pregnancy and nutritional status of the individual. Pregnancy has generally been reported to cause a depression of hepatic monooxygenase activities. Low-protein diets and protein-energy malnutrition have also been associated with a reduced activity of monooxygenases in nonpregnant animals. We investigated the combined effects of pregnancy and protein-energy malnutrition on liver monooxygenase O-dealkylation activity. On pregnancy day 0 rats were assigned at random to a group fed ad libitum (well-nourished, WN) or to a malnourished group (MN) which received half of the WN food intake (12 g/day). WN and MN rats were killed on days 0 (nonpregnant), 11 or 20 of pregnancy and ethoxy- (EROD), methoxy- (MROD) and penthoxy- (PROD) resorufin O-dealkylation activities were measured in liver microsomes. Only minor changes in enzyme activities were observed on pregnancy day 11, but a clear-cut reduction of monooxygenase activities (pmol resorufin min-1 mg protein-1) was noted near term (day 0 vs 20, means +/- SD, Student t-test, P<0.05) in WN (EROD: 78.9 +/- 15.1 vs 54.6 +/- 10.2; MROD: 67.8 +/- 10.0 vs 40.9 +/- 7.2; PROD: 6.6 +/- 0. 9 vs 4.3 +/- 0.8) and in MN (EROD: 89.2 +/- 23.9 vs 46.9 +/- 15.0; MROD: 66.8 +/- 13.8 vs 27.9 +/- 4.4; PROD: 6.3 +/- 1.0 vs 4.1 +/- 0. 6) dams. On pregnancy day 20 MROD was lower in MN than in WN dams. Malnutrition did not increase the pregnancy-induced reduction of EROD and PROD activities. Thus, the present results suggest that the activities of liver monooxygenases are reduced in near-term pregnancy and that protein-energy malnutrition does not alter EROD or PROD in pregnant rats.

Facebook in educational research: a bibliometric analysis

Facebook has become the object of research in different areas. The present study presents a bibliometric analysis of the scientific literature related to the use of this social network in educational research. To this end, bibliometric techniques were applied in the analysis of scientific articles indexed at the Web of Science Core Collection, from Thomson Reuters, and linked to the research areas of Education/Educational Research. This resulted in the identification of, among others, developments in scientific production, the most important journals that publish papers on the topic, the main authors and the main articles published in the area. The results indicate the growth of scientific production in the area from 2008 onwards, pointing to Computers and Education as the most relevant journal by number of publications (22) and impact factor and indicate that authors from the United States, Australia, Taiwan, United Kingdom and South Africa stand out in the construction of knowledge on educational research applying Facebook. Moreover, the ego-network of the Educational Research area shows that this area coexists with other areas of knowledge in the use of social networking, such as Computer Science, Linguistics and Health Sciences, indicating an interdisciplinary and transversal nature in different areas of research.

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.