Immobilization of Aspergillus ficuum tannase in calcium alginate beads and its application in the treatment of boldo (Peumus boldus) tea.

Tannase (tannin acyl hydrolase, E.C. 3.1.1.20) is an enzyme that catalyzes the hydrolysis of ester and depside linkages in hydrolysable tannins such as tannic acid, releasing gallic acid and glucose. It has several commercial applications in food industry, among which are gallic acid production, reduction of tannin content in fruit juices, and preparation of instantaneous tea. In this study we immobilized Aspergillus ficuum tannase in calcium alginate beads and then used it to treat boldo (Peumus boldus) tea. Such a technique allowed entrapping tannase with a 75% efficiency and appreciably increasing its thermal and pH stability compared with the free enzyme. Storage stability and reuse of the immobilized enzyme were very promising, in that about 60% of starting enzyme activity was retained after bead storage for 90 days at 4 °C or after six cycles of use. Boldo tea treatment with immobilized tannase for 120 min at 40 °C led to 31 and 60% removals of tannins and epigallocatechin gallate, an increase of about two orders of magnitude in gallic acid content, 56 and 109% increases in total flavonoids and epigallocatechin contents, a 42.8% increase in antioxidant activity and significant enhancements of tea color, clarity and pH.

Characterisation of bare and tannase-loaded calcium alginate beads by microscopic, thermogravimetric, FTIR and XRD analyses.

Incorporating enzymes into calcium alginate beads is an effective method to immobilise them and to preserve, at the same time, their catalytic activity. Sodium alginate was mixed with Aspergillus ficuum tannase in aqueous solution, and tannase-loaded calcium alginate beads were prepared using a simple droplet-based microfluidic system. Extensive experimental analysis was carried out to characterise the samples. Microscopic imaging revealed morphological differences between the surfaces of bare alginate matrix and tannase-loaded alginate beads. Thermal analysis allowed assessing the hydration contents of alginate and revealed the presence of tannase entrapped in the loaded beads, which was confirmed by vibrational spectroscopy. X-ray diffraction allowed us to conclude that alginate of tannase-loaded beads is not crystalline, which would make them suitable as carriers for possible controlled release. Moreover, they could be used in food applications to improve tea quality or clarify juices.

Acetylcholinesterase of mangrove oyster Crassostrea rhizophorae: A highly thermostable enzyme with promising features for estuarine biomonitoring.

Enzyme biomarkers from several aquatic organisms have been used for assessing the exposure to contaminants at sublethal levels. Amongst them, the cholinesterases are commonly extracted from several organisms to evaluate/measure organophosphate and carbamate neurotoxic effects. Acetylcholinesterase (AChE; EC 3.1.1.7) is an enzyme of the group of serine esterases that acts on the hydrolysis of the neurotransmitter acetylcholine allowing the intermittence of the nerve impulses responsible for the neuronal communication. This enzyme is the main target for the action of some pesticides and the inhibition of its activity in bivalve mollusks may be used as biomarker due to their filter-feeding habit. In this context, the present study aimed to characterize physicochemical and kinetic parameters of the AChE extracted from gills and viscera of the oyster Crassostrea rhizophorae and investigate the in vitro effect of pesticides (dichlorvos, diazinon, chlorpyrifos, methyl-parathion, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron and novaluron) in search for assessing its potential as biomarker. Specific substrates and inhibitors evidenced the predominance of AChE in both tissues. The optimum pH found for gills and viscera AChE were 8.0 and 8.5, respectively. The maximum peak of activity occurred at 70 °C for gill AChE and 75 °C for viscera AChE. The enzymes of both tissues presented remarkable thermostability. The Michaelis-Menten constant for both enzymes were 1.32 ±â€¯0.20 mM for gills and 0.43 ±â€¯0.12 mM for viscera. The Vmax values for gills and viscera were 53.57 ±â€¯1.72 and 27.71 ±â€¯1.15 mU/mg, respectively. The enzymes were able to reduce the activation energy to 9.75 kcal mol-1 (gills) and 11.87 kcal mol-1 (viscera) obtaining rate enhancements of 3.57 × 105 and 1.01 × 104, respectively, in relation to non-catalyzed reactions. Among the pesticides under study, the carbamates carbaryl and carbofuran exerted the strongest inhibitory effects on the enzyme activity achieving important degrees of inhibition at concentrations below national and international current regulations. The first observation of the effects of benzoylurea pesticides (diflubenzuron and novaluron) on AChE from mollusks is reported here. The gills AChE of C. rhizophorae showed potential to be specific biomarker for the carbamate carbaryl while the viscera AChE showed it for carbofuran. According to their features, these enzymes may be proposed as promising tools for estuarine monitoring as well as biocomponent of biosensor devices.

DNA binding and Topoisomerase inhibition: How can these mechanisms be explored to design more specific anticancer agents?

DNA is considered one of the most promising targets of molecules with anticancer activity potential. Its key role in various cell division mechanisms, which commands the intense multiplication of tumor cells, is considered in studies with compounds whose mechanisms of action suggest likeliness of interaction. In addition, inhibition of enzymes that actively participate in biological functions of cells such as Topoisomerase, is seen as a primary factor for conducting several events that result in cell death. Discovery of new anticancer chemotherapeutical capable of interacting with DNA and inhibiting Topoisomerase enzymes is highlighted in anticancer research. The present review aims at showing through distinct biological tests the performance of different candidates to anticancer drugs and their respective chemical modifications, which are crucial and/or determinant for DNA affinity and inhibition of important enzymes in cells' vital processe to either separately or synergistically optimize anticancer activity.

Trypsin purification using magnetic particles of azocasein-iron composite.

This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources.

Aluminium sulfate exposure: A set of effects on hydrolases from brain, muscle and digestive tract of juvenile Nile tilapia (Oreochromis niloticus).

Aluminium is a major pollutant due to its constant disposal in aquatic environments through anthropogenic activities. The physiological effects of this metal in fish are still scarce in the literature. This study investigated the in vivo and in vitro effects of aluminium sulfate on the activity of enzymes from Nile tilapia (Oreochromis niloticus): brain acetylcholinesterase (AChE), muscle cholinesterases (AChE-like and BChE-like activities), pepsin, trypsin, chymotrypsin and amylase. Fish were in vivo exposed during 14days when the following experimental groups were assayed: control group (CG), exposure to Al2(SO4)3 at 1µg·mL-1 (G1) and 3µg·mL-1 (G3) (concentrations compatible with the use of aluminium sulfate as coagulant in water treatment). In vitro exposure was performed using animals of CG treatment. Both in vivo and in vitro exposure increased cholinesterase activity in relation to controls. The highest cholinesterase activity was observed for muscle BChE-like enzyme in G3. In contrast, the digestive enzymes showed decreased activity in both in vivo and in vitro exposures. The highest inhibitory effect was observed for pepsin activity. The inhibition of serine proteases was also quantitatively analyzed in zymograms using pixel optical densitometry as area under the peaks (AUP) and integrated density (ID). These results suggest that the inhibition of digestive enzymes in combination with activation of cholinesterases in O. niloticus is a set of biochemical effects that evidence the presence of aluminium in the aquatic environment. Moreover, these enzymatic alterations may support further studies on physiological changes in this species with implications for its neurological and digestive metabolisms.

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.

Brain acetylcholinesterase of jaguar cichlid (Parachromis managuensis): From physicochemical and kinetic properties to its potential as biomarker of pesticides and metal ions.

This contribution aimed to characterize physicochemical and kinetic parameters of the brain cholinesterases (ChEs) from Parachromis managuensis and investigate the in vitro effects of pesticides and metal ions on its activity intending to propose as biomarker. This species is suitable for this investigation because (1) it was recently introduced in Brazil becoming invasive (no restrictions on capture) and (2) occupies the top of the food chain (being subject to bioaccumulation). The enzyme extract was exposed to 10 metal ions (Al(3+), Ba(2+), Cd(2+), Cu(2+), Hg(2+), Mg(2+), Mn(2+), Pb(2+), Fe(2+) and Zn(2+)) and ChEs selective inhibitors (BW284c51, Iso-OMPA, neostigmine and serine). The extract was also incubated with organophosphate (dichlorvos) and carbamate pesticides (carbaryl and carbofuran). Inhibition parameters (IC20, IC50 and ki) were determined. Selective inhibitors and kinetic parameters confirmed acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) -like as responsible for the ChE activities, most AChE. The IC50 values for pesticides were: 1.68µM (dichlorvos); 4.35µM (carbaryl) and 0.28µM (carbofuran). Most of the analyzed ions did not show significant effect at 1mM (p=0.05), whereas the following ions inhibited the enzyme activity in the order: Hg(2+)>Cu(2+)>Cd(2+)>Zn(2+). Mercury ion strongly inhibited the enzyme activity (IC20=0.7µM). The results about allow to conclude that P. managuensis brain AChE is a potential biomarker for heavy metals and pesticides under study, mainly for the carbamate carbofuran once it was capable to detect 6-fold lower levels than the limit concentration internationally recommended.

Digestive enzyme activity in the intestine of Nile tilapia (Oreochromis niloticus L.) under pond and cage farming systems.

The effect of different farming systems (cage, pond) upon digestive enzyme activities of Nile tilapia was evaluated. Juvenile Nile tilapia (87.61 ± 1.52 g) were simultaneously cultured in pond and cage systems during 90 days. Cages used nutritional biphasic plan (35 and 32 % crude protein-CP feeds) and ponds used nutritional triphasic plan (35, 32 and 28 % CP feeds). Biometric measurements were monthly performed for adjustments in feeding regimes and removal of intestine tissues to evaluate the performance of enzyme activities. Total proteolytic, amylase and lipase activities were not statistically different between the treatments throughout the periods analyzed (31, 63 and 94 days of culture). However, trypsin and chymotrypsin activities were higher with 31 and 63 days of culture in fish from pond system, suggesting that natural food may have influenced these activities. A positive correlation was observed between the recommended concentration of essential amino acids for Nile tilapia and specific aminopeptidases activity in fish cage system. Substrate-SDS-PAGE revealed 12 active proteolytic bands in both systems. However, integrated density (ID) values were higher in the bands of ponds. Specimens of either cage or pond exhibited five bands of amylolytic activity. Fish from cage and pond systems showed the highest values of ID within 31 days of cultivation. In this study, the complexity of digestive functions could be verified for animals maintained under commercial conditions. Some of the assessed enzymes may show adaptations of their activities and/or expression that allow the fish to achieve a more efficient nutrient assimilation.

Characterization of catalytic efficiency parameters of brain cholinesterases in tropical fish.

Brain cholinesterases from four fish (Arapaima gigas, Colossoma macropomum, Rachycentron canadum and Oreochromis niloticus) were characterized using specific substrates and selective inhibitors. Parameters of catalytic efficiency such as activation energy (AE), k(cat) and k(cat)/k(m) as well as rate enhancements produced by these enzymes were estimated by a method using crude extracts described here. Despite the BChE-like activity, specific substrate kinetic analysis pointed to the existence of only acetylcholinesterase (AChE) in brain of the species studied. Selective inhibition suggests that C. macropomum brain AChE presents atypical activity regarding its behavior in the presence of selective inhibitors. AE data showed that the enzymes increased the rate of reactions up to 10(12) in relation to the uncatalyzed reactions. Zymograms showed the presence of AChE isoforms with molecular weights ranging from 202 to 299 kDa. Values of k(cat) and k(cat)/k(m) were similar to those found in the literature.

Synthesis, DNA binding and topoisomerase I inhibition activity of thiazacridine and imidazacridine derivatives.

Thiazacridine and imidazacridine derivatives have shown promising results as tumors suppressors in some cancer cell lines. For a better understanding of the mechanism of action of these compounds, binding studies of 5-acridin-9-ylmethylidene-3-amino-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-imidazolidin-4-one and 3-acridin-9-ylmethyl-thiazolidin-2,4-dione with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopy and circular dichroism spectroscopy were performed. The binding constants ranged from 1.46 × 10(4) to 6.01 × 10(4) M(-1). UV-Vis, fluorescence and circular dichroism measurements indicated that the compounds interact effectively with ctDNA, both by intercalation or external binding. They demonstrated inhibitory activities to human topoisomerase I, except for 5-acridin-9-ylmethylidene-2-thioxo-1,3-thiazolidin-4-one. These results provide insight into the DNA binding mechanism of imidazacridines and thiazacridines.

Immunochemiluminescent detection of galectin-3 in tumoral tissue from prostate.

This work proposes a chemiluminescent quantitative method for galectin-3 (Gal3) detection in prostate tissues. Monoclonal antibody anti-Gal3 was conjugated to acridinium ester (AE) and the complex formed with Gal3 in the prostate tissue was chemiluminescently detected. The light emission (expressed in Relative Light Unit-RLU) showed mean values higher for benign prostatic hyperplasia than normal tissues and adenocarcinoma. These differences showed to be statistically significant (p < 0.001). There was a linear relationship between RLU and tissue area. Furthermore, these values were dramatically reduced when the tissue samples were previously incubated with non labeled anti-Gal3. Finally, the anti-Gal3-AE solution in buffer stored at 4°C and the treated samples showed to be stable during a year and at least 72 h, respectively. Gal3 content in prostate tissue was higher in benign prostatic hyperplasia than normal tissues and much lower in adenocarcinoma. This quantitative, specific and sensitive method based on labeling antibody to acridinium ester can be applied to detect antigen in tissue.

Kinetic and physicochemical properties of brain acetylcholinesterase from the peacock bass (Cichla ocellaris) and in vitro effect of pesticides and metal ions.

Brain acetylcholinesterase (AChE; EC 3.1.1.7) from peacock bass (Cichla ocellaris) was characterized and the effect of organophosphorus and carbamate pesticides as well as ions and heavy metals was evaluated. The kinetic parameters K(m) and V(max) were determined as 0.769 mM and 0.189 U/mg of protein respectively. Optimal pH and temperature were found to be 8.0 and 45°C. The enzyme retained approximately half of the activity after incubation at 50°C for 30 min. Total cholinesterase activity on brain of this species can be ascribed to AChE according to selective inhibitors analysis (neostigmine, eserine and BW284c5 reduced its activity whereas no effect was noticed for Iso-OMPA). Seven pesticides (five organophosphates: dichlorvos, diazinon, chlorpyrifos, temephos, tetraethyl pyrophosphate - TEPP and two carbamates: carbaryl and carbofuran) showed inhibitory effects on C. ocellaris AChE. However, the strongest effect was observed with carbofuran (IC(50)=0.21 µM and K(i)=2.57 × 10(-3) µM). The following ions (1 mM) showed to inhibit its activity (decrescent order): Hg(2+)>As(3+)>Cu(2+)>Zn(2+). EDTA(2-) did not affect enzyme activity. The present study provides assay conditions and data to suggest this enzyme as in vitro biomarker of organophosphorus and carbamate pesticides in routine environmental screening programs.

Comparative effect of pesticides on brain acetylcholinesterase in tropical fish.

Monitoring of pesticides based on acetylcholinesterase (AChE; EC 3.1.1.7) inhibition in vitro avoids interference of detoxification defenses and bioactivation of some of those compounds in non-target tissues. Moreover, environmental temperature, age and stress are able to affect specific enzyme activities when performing in vivo studies. Few comparative studies have investigated the inter-specific differences in AChE activity in fish. Screening studies allow choosing the suitable species as source of AChE to detect pesticides in a given situation. Brain AChE from the tropical fish: pirarucu (Arapaima gigas), cobia (Rachycentron canadum) and Nile tilapia (Oreochromis niloticus) were characterized and their activities were assayed in the presence of pesticides (the organophosphates: dichlorvos, diazinon, chlorpyrifos, temephos, tetraethyl pyrophosphate- TEPP and the carbamates: carbaryl and carbofuran). Inhibition parameters (IC50 and Ki) for each species were found and compared with commercial AChE from electric eel (Electrophorus electricus). Optimal pH and temperature were found to be 8.0 and 35-45 °C, respectively. A. gigas AChE retained 81% of the activity after incubation at 50 °C for 30 min. The electric eel enzyme was more sensitive to the compounds (mainly carbofuran, IC50 of 5 nM), excepting the one from A. gigas (IC50 of 9 nM) under TEPP inhibition. These results show comparable sensitivity between purified and non-purified enzymes suggesting them as biomarkers for organophosphorus and carbamate detection in routine environmental and food monitoring programs for pesticides.

α-L-rhamnosidase of Aspergillus terreus immobilized on ferromagnetic supports.

α-L-rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/µg of protein) and POS/PVA (0.59 nkat/µg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/µg of protein). The activity-pH and activity-temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40-70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K (m) of α-L-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).

Chemiluminescent detection of carbohydrates in the tumoral breast diseases.

Nowadays, there is an increase of investigations into the fibroadenoma, mainly because some studies have shown that the occurrence of fibroadenoma is linked to an increased risk of developing breast carcinoma. Currently, the chemiluminescence biomarkers are applied for validation methods and screening. Here, a lectin chemiluminescence is proposed as new histochemistry method to identify carbohydrates in mammary tumoral tissues. The lectins concanavalin A (Con A) and peanut agglutinin (PNA) conjugated to acridinium ester were used to characterize the glycocode of breast tissues: normal, fibroadenoma, and invasive duct carcinoma (IDC). The lectin chemiluminescence expressed in relative light units (RLU) was higher in fibroadenoma and IDC than in normal tissue for both lectins tested. The relationship RLU emission versus tissue area described a linear and hyperbolic curve for IDC and fibroadenoma, respectively, using Con A whereas hyperbolic curves for both transformed tissues using PNA. RLU was abolished by inhibiting the interaction between tissues and lectins using their specific carbohydrates: methyl-α-D: -mannoside (Con A) and galactose (PNA). The intrinsic fluorescence emission did not change with combination of the lectins (Con A/PNA) to the acridinium ester for hydrophobic residues. These results represent the lectin chemiluminescence as an alternative of histochemistry method for tumoral diagnosis in the breast.

Characterization of acetylcholinesterase from the brain of the Amazonian tambaqui (Colossoma macropomum) and in vitro effect of organophosphorus and carbamate pesticides.

In the present study, acetylcholinesterase (AChE) from the brain of the Amazonian fish tambaqui (Colossoma macropomum) was partially characterized and its activity was assayed in the presence of five organophosphates (dichlorvos, diazinon, chlorpyrifos, and tetraethyl pyrophosphate [TEPP]) and two carbamates (carbaryl and carbofuran) insecticides. Optimal pH and temperature were 7.0 to 8.0 and 45°C, respectively. The enzyme retained approximately 70% of activity after incubation at 50°C for 30 min. The insecticide concentration capable of inhibiting half of the enzyme activity (IC50) for dichlorvos, chlorpyrifos, and TEPP were calculated as 0.04 µmol/L, 7.6 µmol/L, and 3.7 µmol/L, respectively. Diazinon and temephos did not inhibit the enzyme. The IC50 values for carbaryl and carbofuran were estimated as 33.8 µmol/L and 0.92 µmol/L, respectively. These results suggest that AChE from the juvenile C. macropomum brain could be used as an alternative biocomponent of organophosphorus and carbamate biosensors in routine pesticide screening in the environment.

Immobilization of anti-galectin-3 onto polysiloxane-polyvinyl alcohol disks for tumor prostatic diseases diagnosis.

This work aimed to immobilize the antibody anti-galectin-3 onto polysiloxane-polyvinyl alcohol (POS-PVA) support, to evaluate its capacity to capture the serum antigen galectin-3 and to quantify by ELISA the antigen levels in sera from patients with prostatic adenocarcinoma (PA) and benign prostatic hyperplasia (BPH) and healthy individuals. Also, for comparative effect, the galectin-3 expression in the prostate tissue through immunohistochemistry was evaluated. The optical density (galectin-3 level) values established for the sera from PA and BPH patients were lower compared with those found for the healthy individuals. Galectin-3 immunohistochemically showed a significant increase and reduction of the cytoplasmatic protein expression in BPH and PA, respectively, compared with the normal prostate. These results showed that POS-PVA disks could be used as solid phase to immobilize serum galectins and in immunoassays procedures for the correspondent IgG anti-galectins detection in human sera.

Effect of dichlorvos on the acetylcholinesterase from tambaqui (Colossoma macropomum) brain.

Dichlorvos is an acutely toxic organophosphorous pesticide that is known as a classical acetylcholinesterase (AChE; EC 3.1.1.7) inhibitor. Here, the brain AChE from the important Amazonian fish tambaqui (Colossoma macropomum) was assayed in the presence of this insecticide and also of deltamethrin, a classical sodium and potassium channel inhibitor (negative control). Four tissue homogenates were analyzed in triplicate for AChE activity using acetylthiocholine as the substrate and 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as the color-developing agent. Each tissue homogenate represented pooled brains from five fish. The inhibitory effect of dichlorvos on AChE activities was determined at concentrations from 0.001 to 10 ppm and compared to controls. This effect followed an exponential decay model (y = 9.420 + 26.192e(-x/5.380); r2 = 0.989), presenting IC50 (the concentration of dichlorvos that is required for 50% of AChE inhibition) of 0.081 ppm (0.368 micromol/L). No effect was observed for the deltamethrin, and the concentration 0.0452 micromol/L of dichlorvos was significantly different from this control. These results suggest that tambaqui brain AChE can be proposed as a biomarker for dichlorvos and can be used as a tool for aquatic environment monitoring.

Prevalence of toxocariasis in northeastern Brazil based on serology using recombinant Toxocara canis antigen.

To evaluate the prevalence of toxocariasis in children in Jaboatão dos Guararapes, Pernambuco in northeastern Brazil, 215 serum samples were examined by an enzyme-linked immunosorbent assay (ELISA) using a recombinant Toxocara canis antigen. In the ELISA, 26 (12.1%) of 215 subjects were positive. In a dot-blot assay using 53 of 215 serum samples, the diagnostic results correlated with those obtained by the ELISA. Moreover, it has been confirmed that the recombinant T. canis antigen was highly specific for toxocariasis by ELISA using serum samples positive for antibody to Ascaris lumbricoides. Considering the specificity of the recombinant antigen to toxocariasis, the ELISA or dot-blot assay using the recombinant T. canis antigen is recommended in tropical and sub-tropical regions where various parasitic infections are commonly endemic.