Detection of carbamate pesticides in vegetable samples using cholinesterase-based biosensors

Biosensors for determination of carbamates in vegetables based on five different cholinesterases as biorecognition elements and a screen-printed electrode system as an amperometric transducer were developed. Measurements were simply performed by dropping solutions (either sample or substrate) directly on the biosensor. The response of biosensors to selected carbamates (aldicarb, carbaryl, carbofuran, methomyl and propoxur) was characterized. The performance was evaluated on extracts of potatoes and carrots, the results from the AChE biosensor and a standard HPLC procedure were compared. Finally, the biosensor was used for the direct analysis of vegetable juices without any pretreatment steps. In this case, 10 mu g/L levels of added carbofuran and propoxur were reliably identified. The whole procedure takes less than 20 min including 10 min incubation with samples. The concentrations of carbamates determined with biosensor agreed well for carbofuran. Lower response was observed for propoxur.

New selective acetylcholinesterase inhibitors designed from natural piperidine alkaloids

Five new piperidine alkaloids were designed from natural (-)-3-O-acetyl-spectaline and (-)-spectaline that were obtained from the flowers of Senna spectabilis (sin. Cassia spectabilis, Leguminosae). Two semi-synthetic analogues (7 and 9) inhibited rat brain acetylcholinesterase, showing IC50 of 7.32 and 15.1 mu M, and were 21 and 9.5 times less potent against rat brain butyrylcholinesterase, respectively. Compound 9 (1 mg/kg, ip) was fully efficacious in reverting scopolamine-induced amnesia in mice. The two active compounds (7 and 9) did not show overt toxic effects at the doses tested in vivo. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular models of cyclin-dependent kinase 1 complexed with inhibitors

Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDKI and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors. (C) 2004 Elsevier B.V. All rights reserved.

Cholinesterase measurements with an automated kit

Background the Test-mate kit determines acetylcholinesterase (AChE, EC 3.1.1.7) and hemoglobin content of a drop of blood, displaying enzyme activities normalized to 25degreesC. Previous models produced inconsistent results at different temperatures. This report focuses on the current model, ChE 400, and two instruments of a previous OP model.Methods AChE activities were determined by the Ellman assay, using the three kits and a 96-well microplate reader Temperatures ranged from 10 to 37degreesC. Fetal bovine serum was the source of AChE.Results Normalized activities decreased below 20degreesC in the ChE model and below 25 C in the OP models. Activities of the same serum sample differed between the three Test-mate kits, ranging from 1.03 to 1.49 mumoles/min/ml. Percent errors were greater than with the microplate reader at all temperatures.Conclusions Neither we nor the manufacturer recommend the current Test-mate model for fieldwork. Nevertheless, there have been field measurements with Test-Mate kits, and we recommend that an enzyme activity standard be run in parallel with their use. (C) 2002 Wiley-Liss, Inc.

Indole glucoalkaloids from Chimarrhis turbinata and their evaluation as antioxidant agents and acetylcholinesterase inhibitors

As part of our study on bioactive agents from Brazilian rainforest plants, two new glucoalkaloids, 3,4-dehydro-strictosidine (1) and 3,4-dehydro-strictosidinic acid (2), were isolated from Chimarrhis turbinata, along with seven known glucoalkaloids, cordifoline (3), strictosidinic acid (4), strictosidine (5), 5alpha-carboxystrictosidine (6), turbinatine (7), desoxycordifoline (8), and harman-3-carboxylic acid (9). The structures of the new alkaloids were established on the basis of comprehensive spectral analysis, mainly 1D and 2D NMR experiments, as well as high-resolution HRESIMS. Alkaloid 3 showed strong free-radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) as well as pronounced antioxidant activity evidenced by redox properties measured by ElCD-HPLC. Additionally, alkaloids 1-9 were submitted to TLC screening for acetylcholinesterase inhibitors. Both 7 and 8 were shown to be moderate acetylcholinesterase inhibitors at a concentration of 0.1 and 1.0 muM, respectively. In an in vitro rat brain assay, 7 showed moderate activity (IC50 1.86 muM), compared to the standard compound, galanthamine (IC50 0.92 muM).

Structural Basis for Interaction of Inhibitors with Cyclin-Dependent Kinase 2

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

Determination of carbamate residues in crop samples by cholinesterase-based biosensors and chromatographic techniques

An amperometric biosensor based on cholinesterase (ChE) has been used for the determination of selected carbamate insecticides in vegetable samples. The linear range of the biosensor for the N-methylcarbamates (aldicarb, carbaryl, carbofuran, methomyl and propoxur) varied from 5 x 10(-5) to 50 mg kg(-1). Limits of detection were calculated on the basis that the ChE enzymes were 10% inhibited and varied, depending of the combination ChE (as acetyl- or butyrylcholinesterase) vs. inhibitor (pesticide), from 1 x 10(-4) to 3.5 mg kg(-1). The biosensor-based carbamate determination was compared to liquid chromatography/UV methods. Three vegetable samples were spiked with carbofuran and propoxur at 125 mu g kg(-1) followed by conventional procedures. Good correlations were observed for carbofuran in the vegetable extracts (79, 96 and 91% recoveries for potato, carrot and sweet pepper, respectively), whereas for propoxur unsatisfactory results were obtained. Potato and carrot samples were spiked with 10, 50 and 125 mu g kg(-1) carbofuran, followed by direct determination by the amperometric biosensor. The fortified sampler; resulted in very high inhibition values, and recoveries were: 28, 34 and 99% for potato, and 140, 90 and 101% for carrot, respectively, at these three fortification levels. (C) 1998 Elsevier B.V. B.V.

4 '-Aminochalcones As Novel Inhibitors of the Chlorinating Activity of Myeloperoxidase

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

Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Natural Products from Garcinia brasiliensis as Leishmania Protease Inhibitors

The infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which cause renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro Leishmania protease inhibition activity of extracts (hexanic, ethyl-acetate, and ethanolic) and fukugetin, a bioflavonoid purified from the ethyl-acetate extract of the pericarp of the fruit of Garcinia brasiliensis, a tree native to Brazilian forests. The isolated compound was characterized by using spectral analyses with nuclear magnetic resonance, mass spectroscopy, ultraviolet, and infrared techniques. The ethyl-acetate extract and the compound fukugetin showed significant activity as inhibitors of Leishmania's proteases, with mean (+/- SD) IC(50) (50% inhibition concentration of protease activity) values of 15.0 +/- 1.3 mu g/mL and 3.2 +/- 0.5 mu M/mL, respectively, characterizing a bioguided assay. In addition, this isolated compound showed no activity against promastigote and amastigote forms of L. (L.) amazonensis and mammalian cells. These results suggest that fukugetin is a potent protease inhibitor of L. (L.) amazonensis and does not cause toxicity in mammalian or Leishmania cells in vitro. This study provides new perspectives on the development of novel drugs that have leishmanicidal activity obtained from natural products and that target the parasite's proteases.

Heat treatment and inactivation of trypsin-chymotrypsin inhibitors and lectins from beans (Phaseolus vulgaris L.)

This work investigates some factors affecting the inactivation of common bean trypsin inhibitor and phytohemagglutin. Trypsin inhibitor activity was totally stable to heat treatment (30 min, 97C) in the total protein extract, albumin or globulin fraction. Heat treatment of the whole beans easily inactivated the inhibitor. Heat resistance of trypsin inhibitor was intermediate in the bean flour which received the same heat treatment. Independent of sample, the inhibitor was very stable to heat treatment at neutral and acidic pH and labile under strong alkaline conditions. Heating for 30 min in boiling water at pH 12 resulted in complete inactivation of the trypsin inhibitor. Autoclaving (121C) soaked whole beans and flour for 5 min inactivated 55% of the trypsin inhibitor activity in the soaked flour and 75% in the whole beans. After autoclaving 20 min, inactivation of trypsin inhibitor was about 65% in the flour and 80% in the whole beans. The phytohemagglutinin (lectin) activity was totally destroyed in the autoclaved beans after 5 min and in the flour after 15 min.