Erythrocyte acetylcholinesterase as biomarker of pesticide exposure: new and forgotten insights.

Acetylcholinesterase (AChE) acts on the hydrolysis of acetylcholine, rapidly removing this neurotransmitter at cholinergic synapses and neuromuscular junctions as well as in neuronal growth and differentiation, modulation of cell adhesion ("electrotactins") and aryl-acylamidase activity (AAA). This enzyme is also found in erythrocyte, as 160 kDa dimer that anchors to the plasma membrane via glycophosphatidylinositol. The function of this enzyme in erythrocytes has not yet been elucidated; however, it is suspected to participate in cell-to-cell interactions. Here, a review on erythrocyte AChE characteristics and use as biomarker for organophosphorus and carbamate insecticides is presented since it is the first specific target/barrier of the action of these pesticides, besides plasma butyrylcholinesterase (BChE). However, some past and current methods have disadvantages: (a) not discriminating the activities of AChE and BChE; (b) low accuracy due to interference of hemoglobin in whole blood samples. On the other hand, extraction methods of hemoglobin-free erythrocyte AChE allows: (a) the freezing and transporting of samples; (b) samples free of colorimetric interference; (c) data from only erythrocyte AChE activity; (d) erythrocyte AChE specific activity presents higher correlation with the central nervous system AChE than other peripheral ChEs; (e) slow spontaneous regeneration against anti-ChEs agents of AChE in comparison to BChE, thus increasing the chances of detecting such compounds following longer interval after exposure. As monitoring perspectives, hemoglobin-free methodologies may be promising alternatives to assess the degree of exposure since they are not influenced by this interfering agent.

Quantum dot-Cramoll lectin as novel conjugates to glycobiology.

The optical properties of quantum dots (QDs) make them useful tools for biology, especially when combined with biomolecules such as lectins. QDs conjugated to lectins can be used as nanoprobes for carbohydrate expression analysis, which can provide valuable information about glycosylation changes related to cancer and pathogenicity of microorganisms, for example. In this study, we evaluated the best strategy to conjugate Cramoll lectin to QDs and used the fluorescent labeling of Candida albicans cells as a proof-of-concept. Cramoll is a mannose/glucose-binding lectin with unique biological properties such as immunomodulatory, antiparasitic, and antitumor activities. We probed covalent coupling and adsorption as conjugation strategies at different pH values. QDs conjugated to Cramoll at pH7.0 showed the best labeling efficiency in the fluorescence microscopy analysis. Moreover, QD-Cramoll conjugates remained brightly fluorescent and preserved identical biological activity according to hemagglutination assays. Flow cytometry revealed that approximately 17% of C. albicans cells were labeled after incubation with covalent conjugates, while approximately 92% of cells were labeled by adsorption conjugates (both at pH7.0). Inhibition assays confirmed QD-Cramoll specificity, which reduced the labeling to at most 3%. Therefore, the conjugates obtained by adsorption (pH7.0) proved to be promising and versatile fluorescent tools for glycobiology.

Highly fluorescent and superparamagnetic nanosystem for biomedical applications.

This work reports on highly fluorescent and superparamagnetic bimodal nanoparticles (BNPs) obtained by a simple and efficient method as probes for fluorescence analysis and/or contrast agents for MRI. These promising BNPs with small dimensions (ca. 17 nm) consist of superparamagnetic iron oxide nanoparticles (SPIONs) covalently bound with CdTe quantum dots (ca. 3 nm). The chemical structure of the magnetic part of BNPs is predominantly magnetite, with minor goethite and maghemite contributions, as shown by Mössbauer spectroscopy, which is compatible with the x-ray diffraction data. Their size evaluation by different techniques showed that the SPION derivatization process, in order to produce the BNPs, does not lead to a large size increase. The BNPs saturation magnetization, when corrected for the organic content of the sample, is ca. 68 emu g-1, which is only slightly reduced relative to the bare nanoparticles. This indicates that the SPION surface functionalization does not change considerably the magnetic properties. The BNP aqueous suspensions presented stability, high fluorescence, high relaxivity ratio (r 2/r 1 equal to 25) and labeled efficiently HeLa cells as can be seen by fluorescence analysis. These BNP properties point to their applications as fluorescent probes as well as negative T 2-weighted MRI contrast agents. Moreover, their potential magnetic response could also be used for fast bioseparation applications.

High sucrolytic activity by invertase immobilized onto magnetic diatomaceous earth nanoparticles.

Invertase immobilized on magnetic diatomaceous earth nanoparticles (mDE-APTES-invertase) with high sucrolytic activity was obtained by an easy and low-cost method. An experimental design was carried out to investigate the best immobilization conditions and it allowed obtaining an immobilized derivative with a residual specific activity equal to 92.5%. Then, a second experimental design selected the mDE-APTES-invertase with higher specific activity in relation to other derivatives reported in the literature (2.42-fold). Thermal and storage stability for immobilized invertase were found to be 35 °C for 60 min (85% retained activity) and 120 days storage period (80% retained activity), respectively. Besides, a residual activity higher than 60% and 50% were observed for mDE-APTES-invertase after reuse in short and long term, respectively. Given the simple and efficient method to obtain an immobilized derivative with high activity, the mDE nanoparticles appear to be a promising matrix for invertase immobilization as well as for other biomolecules.

CdTe quantum dots conjugated to concanavalin A as potential fluorescent molecular probes for saccharides detection in Candida albicans.

Semiconductor colloidal quantum dots (QDs) have been applied in biological analysis due to their unique optical properties and their versatility to be conjugated to biomolecules, such as lectins and antibodies, acquiring specificity to label a variety of targets. Concanavalin A (Con A) lectin binds specifically to α-d-mannose and α-d-glucose regions of saccharides that are usually expressed on membranes of mammalian cells and on cell walls of microbials. Candida albicans is the most common fungal opportunistic pathogen present in humans. Therefore, in this work, this fungus was chosen as a model for understanding cells and biofilm-forming organisms. Here, we report an efficient bioconjugation process to bind CdTe (Cadmium Telluride) QDs to Con A, and applied the bioconjugates to label saccharide structures on the cellular surface of C. albicans suspensions and biofilms. By accomplishing hemagglutination experiments and circular dichroism, we observed that the Con A structure and biochemical properties were preserved after the bioconjugation. Fluorescence microscopy images of yeasts and hyphae cells, as well as biofilms, incubated with QDs-(Con A) showed a bright orange fluorescence profile, indicating that the cell walls were specifically labeled. Furthermore, flow cytometry measurements confirmed that over 93% of the yeast cells were successfully labeled by QD-(Con A) complex. In contrast, non-conjugated QDs or QDs-(inhibited Con A) do not label any kind of biological system tested, indicating that the bioconjugation was specific and efficient. The staining pattern of the cells and biofilms demonstrate that QDs were effectively bioconjugated to Con A with specific labeling of saccharide-rich structures on C. albicans. Consequently, this work opens new possibilities to monitor glucose and mannose molecules through fluorescence techniques, which can help to optimize phototherapy protocols for this kind of fungus.

Evaluation of glycophenotype in breast cancer by quantum dot-lectin histochemistry.

Cell surface glycoconjugates play an important role in differentiation/dedifferentiation processes and lectins are employed to evaluate them by several methodologies. Fluorescent probes are considered a valuable tool because of their ability to provide a particular view, and are more detailed and sensitive in terms of cell structure and molecular content. The aim of this study was to evaluate and compare the expression and distribution of glycoconjugates in normal human breast tissue, and benign (fibroadenoma), and malignantly transformed (invasive ductal carcinoma) breast tissues. For this, we used mercaptosuccinic acid-coated Cadmium Telluride (CdTe) quantum dots (QDs) conjugated with concanavalin A (Con A) or Ulex europaeus agglutinin I (UEA I) lectins to detect α-D-glucose/mannose and L-fucose residues, respectively. The QD-lectin conjugates were evaluated by hemagglutination activity tests and carbohydrate inhibition assays, and were found to remain functional, keeping their fluorescent properties and carbohydrate recognition ability. Fluorescence images showed that different regions of breast tissue expressed particular types of carbohydrates. While the stroma was preferentially and intensely stained by QD-Con A, ductal cells were preferentially labeled by QD-UEA I. These results indicate that QD-lectin conjugates can be used as molecular probes and can help to elucidate the glycoconjugate profile in biological processes.

Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization.

BACKGROUND: Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish. RESULTS: A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. Km, kcat, and kcat/Km values using BApNA as substrate were 0.689 mM, 6.9 s-1, and 10 s-1 mM-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd2+, Al3+, Zn2+, Cu2+, Pb2+, and Hg2+ at 1 mM, revealing high sensitivity of the enzyme to metal ions. CONCLUSIONS: Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.

Shrimp waste extract and astaxanthin: rat alveolar macrophage, oxidative stress and inflammation.

UNLABELLED: Astaxanthin is a carotenoid known to have antioxidant and antiinflammatory properties. This study examined if shrimp astaxanthin modulates the production of superoxide (O(-)(2)), nitric oxide (NO), and tumor necrosis factor-α (TNF-α) in rat alveolar macrophages. The oxidative effect was induced by phorbol myristate acetate and lipopolysacharide. The treatment was compared with superoxide dismutase, butylated hydroxytoluene, commercial astaxanthin, N-nitric-L-arginine methyl ester and L- canavanine, all administered as a 43.5-µg/mL dose in the presence of 1% EtOH/0.5% DMSO. All treatments maintained cell viability, as observed in the MTT assay, and shrimp extract increased the viable alveolar macrophages to 168%. Shrimp extract and commercial astaxanthin showed a suppressive effect on the generation of both free radicals O(-)(2) and NO, while purified shrimp astaxanthin was specific to NO. TNF-α secretion was correlated with NO production. However, in this correlation, the shrimp extract completely inhibited TNF-α. In the light of these findings, the antioxidant action demonstrated in this study suggests that the shrimp extract could be considered as a promising source of bioactive substances with antioxidant and anti-inflammatory activity. PRACTICAL APPLICATION: The hydrolysis process of shrimp waste generates bioactive products that add economic value to shrimp processing, mainly because they may have applications in nutraceutical and animal feed industry.

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum).

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60 degrees C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.

Trypsin from the processing waste of the lane snapper (Lutjanus synagris) and its compatibility with oxidants, surfactants and commercial detergents.

A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.

ConA and UEA-I lectin histochemistry of parotid gland mucoepidermoid carcinoma.

Mucoepidermoid carcinoma (MEC) corresponds to 5-12% of all salivary gland tumours, and is classified as low, intermediate or high grade. Traditionally, immunohistochemistry was considered as the complementary tool for diagnosis of salivary gland neoplasia. Lectin histochemistry has also been increasingly used in recent years. In this work, lectins were used as histochemical markers for normal and transformed parotid glands. Biopsy specimens of 15 cases diagnosed as MEC (low, intermediate and high grade) of the parotid gland were trypsin- and methanol-H(2)O(2)-treated and incubated with horseradish peroxidase (HRP)-conjugated lectins, Concanavalin A (Con A-HRP) and Ulex europeus I (UEA-I-HRP). Con A stained the neoplasic cells of MEC (all grades). In high and intermediate cases, ductal cells were weakly stained by Con A. UEA-I weakly stained normal cells of the excretory duct and neoplasic cells in high grade. Neoplasic cells in intermediate grade were moderately stained and in low grade, the cell membrane was intensely stained with UEA-I. Stroma presented a direct relation between malignancy and staining intensity for UEA-I. The results indicated that lectin histochemistry distinguished the cell biology among histological grades of MEC.

Ferromagnetic levan composite: an affinity matrix to purify lectin.

A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.

Lectin staining patterns in human gastric mucosae with and without exposure to Helicobacter pylori.

The aim of the present study was to evaluate qualitative changes in the glycoconjugate expression in human gastric tissue of positive and negative patients for Helicobacter pylori, through lectins: Wheat Germ Agglutinin (WGA) and Concanavalin A (Con A). The lectins recognized differently the glycoconjugates in the superficial mucous layer at the gastric tissues. The results suggest a significant change in the carbohydrate moieties present on the surface of the gastric cells during infection.

Detection of S100 protein from prostatic cancer patients using anti-S100 protein antibody immobilized on POS-PVA discs.

The S100 proteins have been extensively used as cancer biomarkers. The objectives of the present work were to immobilize the antibody anti-protein S100 to a net of semi-interpenetrated of polysiloxane and polyvinyl alcohol (POS-PVA discs), to investigate its capacity to capture S100 protein from serum and to quantify it by ELISA in sera from patients with prostatic adenocarcinoma (n = 15) and healthy individuals (n = 10). Also these values were compared to the S100 protein expression in the prostatic tissue through immunohistochemistry. The POS-PVA discs fixed about 92.8% of the offered antibody (7.75 microg of antibody per disc). The best values of the immobilized no-marked antibody anti-S100 and serum dilution were found to be 10 microg and 1:400, respectively. Optical density (OD) values for the sera of patients (0.425 +/- 0.042) with prostatic adenocarcinoma were significantly lower (P < 0.05) compared to those established for the healthy individuals (1.034 +/- 0.124). In the immunohistochemistry study no significant variations were observed in the number of positive S100 cells between prostatic adenocarcinoma (153.45 +/- 16.82) and normal prostate (147.04 +/- 18.98). These results showed a clear difference between S100 proteins expressed in tissue and presented in serum during the prostatic tissue neoplasic transformation. Sera analysis was more sensitive than immunohistochemistry S100 protein detection in the prostate tissue besides the advantage to be less invasive method.

Langerhans cells in cutaneous tumours: immunohistochemistry study using a computer image analysis system.

Immunohistochemistry, based on antibody anti-S100 protein, was used to evaluate the Langerhans cells (LC) in benign and malign skin neoplasias. These cells were quantitatively estimated using a computer image analysis (OPTIMAS software system, Version 6.1) in skin biopsies diagnosed as basal cell carcinoma (BCC), epidermoid carcinoma (EpC), trichoepithelioma (TE), keratoacanthoma (KA), seborreic keratosis (SK) and actinic keratosis (AK). The antibody anti-S100 protein recognized them. No significant variations were observed in the number of LC among malignant tumour (BCC = 23.25 +/- 5.81 and EpC = 20.88 +/- 4.24). Benign lesions (AK = 33.04 +/- 7.11; TE = 55.74 +/- 9.35; SK = 42.38 +/- 9.92, and KA = 47.62 +/- 10.4) presented a higher number of LC when they were compared among them and to malignant and normal tissues. No significant differences were observed in LC area and volume between benign and malign neoplasias. These results indicate possibly differences in the immunogenicity between benign and malign epidermic tumours. In conclusion, the experimental computer assessment method was reliable and consistent to morphometric analysis of tumoural tissues.

Genetic characterization of Entamoeba dispar isolates in Northeast Brazil.

The genetic variability of Entamoeba dispar strains was investigated in 39 positive isolates on a survey of 1783 individuals from two different cities of Northeast Brazil (Recife and Macaparana) using two polymorphic species-specific loci (loci 1-2 and 5-6). A combinatory clustering analysis revealed no geographical correlation and remarkable genetic polymorphism among all the isolates examined. Nevertheless, a comparison of the frequency of eight individual PCR products, shared by both Recife and Macaparana populations, for the two loci, showed that only one product of locus 5-6 was significantly different between the two cities. These results suggested that the Macaparana population is infected by similar strains and that locus 5-6 shows potential in assaying questions related to the molecular epidemiology of this region.

Determination of the prevalence of Entamoeba histolytica and E. dispar in the pernambuco state of northeastern Brazil by a polymerase chain reaction.

Previous studies using methods varying from traditional serologic tests to molecular biology techniques have shown that in northeastern Brazil, Entamoeba dispar was more prevalent than E. histolytica. In this study, the prevalence was established by using E. histolytica stool antigen detection kits and a polymerase chain reaction (PCR) with genomic DNA extracted from cultured trophozoites in all four-nuclei, amoeba-positive samples from a population living in Macaparana in northeastern Brazil. Among 1,437 stool samples analyzed, only 59 (4.1%) were positive for four nuclei amoeba. However, all of these samples were negative in an immunoenzymatic assay for the presence of E. histolytica-specific galactose adhesin. Of 59 cultivated samples, only 31 showed trophozoites. Extraction of DNA from these 31 samples, followed by the PCR, showed that 23 samples (74.19%) were positive for E. dispar and no amplification was observed for pathogenic E. histolytica. The remaining eight samples were negative for both species. These findings are consistent with those previously reported.

Prevalence of IgG antibodies specific to Toxoplasma gondii among blood donors in Recife, Northeast Brazil.

A serological survey of Toxoplasma gondii infection in blood donors was carried out in order to identify seroprevalence in Recife, Brazil. Sera from 160 individuals (119 male and 41 female) were evaluated by using a Toxoplasma IgG-antibody enzyme immunoassay (Denka Seiken Co., LTD., Tokyo, Japan). The seropositive percentual for males (79.0%) showed to be higher (p < 0.05) than for females (63.4%). This percentage increases with age, ranging from 18.2% to 92.6% for individuals aging under 20 and 40-50 years old, respectively. For women of childbearing age (18-40 years) it was found a prevalence of 51.6%.