Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101 - to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays. (C) 2009 Elsevier Inc. All rights reserved.

Influence of gamma radiation onto polymeric matrix with papain

Papain is a proteolytic enzyme that has been widely used as debridement agent for scars and wound healing treatment. However, papain presents low stability, which limits its use to extemporaneous or short shelf-life formulations. The purpose of this study was to entrap papain into a polymeric matrix in order to obtain a drug delivery system that could be used as medical device. Since these systems must be sterile, gamma radiation is an interesting option and presents advantages in relation to conventional agents: no radioactive residues are formed: the product can be sterilized inside the final packaging and has an excellent reliability. The normative reference for the establishment of the sterilizing dose determines 25 kGy as the inactivation dose for viable microorganisms. A silicone dispersion was selected to prepare membranes containing 2% (w/w) papain. Irradiated and non-irradiated membranes were simultaneously assessed in order to verify whether gamma radiation interferes with the drug-releasing profile. Results showed that irradiation does not affect significantly papain release and its activity. Therefore papain shows radioresistance in the irradiation conditions applied. In conclusion, gamma radiation can be easily used as sterilizing agent without affecting the papain release profile and its activity onto the biocompatible device is studied. (C) 2009 Elsevier Ltd. All rights reserved.

3D-Pharmacophore mapping of thymidine-based inhibitors of TMPK as potential antituberculosis agents

Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis monophosphate kinase (TMPKmt) is essential to DNA replication. Thus, this enzyme represents a promising target for developing new drugs against TB. In the present study, the receptor-independent, RI, 4D-QSAR method has been used to develop QSAR models and corresponding 3D-pharmacophores for a set of 81 thymidine analogues, and two corresponding subsets, reported as inhibitors of TMPKmt. The resulting optimized models are not only statistically significant with r (2) ranging from 0.83 to 0.92 and q (2) from 0.78 to 0.88, but also are robustly predictive based on test set predictions. The most and the least potent inhibitors in their respective postulated active conformations, derived from each of the models, were docked in the active site of the TMPKmt crystal structure. There is a solid consistency between the 3D-pharmacophore sites defined by the QSAR models and interactions with binding site residues. Moreover, the QSAR models provide insights regarding a probable mechanism of action of the analogues.

Rational Design and 3D-Pharmacophore Mapping of 5 `-Thiourea-Substituted alpha-Thymidine Analogues as Mycobacterial TMPK Inhibitors

Thymidine monophosphate kinase (TMPK) has emerged as an attractive target for developing inhibitors of Mycobacterium tuberculosis growth. In this study the receptor-independent (RI) 4D-QSAR formalism has been used to develop QSAR models and corresponding 3D-pharmacophores for a set of 5`-thiourea-substituted alpha-thymidine inhibitors. Models were developed for the entire training set and for a subset of the training set consisting of the most potent inhibitors. The optimized (RI) 4D-QSAR models are statistically significant (r(2) = 0.90, q(2) = 0.83 entire set, r(2) = 0.86, q(2) = 0.80 high potency subset) and also possess good predictivity based on test set predictions. The most and least potent inhibitors, in their respective postulated active conformations derived from the models, were docked in the active site of the TMPK crystallographic structure. There is a solid consistency between the 3D-pharmacophore sites defined by the QSAR models and interactions with binding site residues. This model identifies new regions of the inhibitors that contain pharmacophore sites, such as the sugar-pyrimidine ring structure and the region of the 5`-arylthiourea moiety. These new regions of the ligands can be further explored and possibly exploited to identify new, novel, and, perhaps, better antituberculosis inhibitors of TMPKmt. Furthermore, the 3D-pharmacophores defined by these models can be used as a starting point for future receptor-dependent antituberculosis drug design as well as to elucidate candidate sites for substituent addition to optimize ADMET properties of analog inhibitors.

Cytotoxicity and Endothelial Cell Adhesion of Lyophilized and Irradiated Bovine Pericardium Modified With Silk Fibroin and Chitosan

Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.

Assessment of Ametryn Contamination in River Water, River Sediment, and Mollusk Bivalves in Sao Paulo State, Brazil

Sao Paulo state, Brazil, is one of the main areas of sugar cane agriculture in the world. Herbicides, in particular, ametryn, are extensively used in this extensive area, which implies that this herbicide is present in the environment and can contaminate the surface water by running off. Thereby, residues of ametryn were analyzed in samples of river water an river sediment and in freshwater bivalves obtained from the rivers Sapucai, Pardo and Mogi-Guacu in Sao Paulo State, Brazil. Samples were taken in the winter of 2003 and 2004 in two locations in each river. The specimens of freshwater bivalves collected and analyzed were Corbicula fluminea, an exotic species, and Diplodon fontaineanus, a native species. Additionally, the evaluation of the ability of bioconcentration and depuration of ametryn by the freshwater bivalve Corbicula fluminea was also performed. Ametryn concentrations in the samples were measured by liquid chromatography coupled to mass spectrometry. Residues of ametryn in water (50 ng/L) and in freshwater bivalves (2-7 ng/g) were found in the Mogi-Guacu River in 2004, and residues in river sediments were found in all rivers in 2003 and 2004 (0.5-2 ng/g). The observation of the aquatic environment through the analysis of these matrixes, water, sediment, and bivalves, revealed the importance of the river sediment in the accumulation of the herbicide ametryn, which can contaminate the biota.

Electrospray MS-based characterization of beta-carbolines - mutagenic constituents of thermally processed meat

The beta-carbolines 1-methyl-9H-pyrido [3,4-b]indole and 9H-pyrido[3,4b]indole have been implicated as having causative roles in a number of human diseases, such as Parkinson`s disease and cancer. As they can be formed during the heating of protein-rich food, a number of analytical methodologies have been proposed for their detection and quantification in foodstuff For this purpose, LC-MS and LC-MS/MS have emerged as the most specific analytical methods, and the quantification is based on the occurrence of unusual ions, such as [M+H-(H(center dot))](+) and [M + H-2H](+). In this study, we have investigated the formation of these ions by accurate-mass electrospray MS/MS and demonstrated that these ions are formed from gas-phase ion-molecule reactions between water vapor present in the collision cell and the protonated molecule of 1-methyl-9H-pyrido [3,4-b]indole and 9H-pyrido[3,4b]indole. Although this reaction has been previously described for heterocyclic amine ions, it has been overlooked in the most of recent LC-MS and LC-MS/MS studies, and no complete data of the fragmentation are reported. Our results demonstrate that additional attention should be given with respect to eliminating water vapor residues in the mass spectrometer when analysis of beta-carbolines is performed, as this residue may affect the reliability in the results of quantification.

Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

Purification and characterization of Ts15, the first member of a new alpha-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: alpha-, beta-, gamma- and kappa-KTx. These peptides display varying selectivity and affinity for K(v) channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Nay channels (Na(V)1.4; Na(V)1.5; Na(V)1.6; Na(V)1.8 and DmNa(V)1) and 12 different subtypes of Kv channels (K(V)1.1 - K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is crosslinked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K(V)1.2 and K(V)1.3 channels with an IC(50) value of 196 +/- 25 and 508 +/- 67 nM, respectively. No effect on Na(V) channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new alpha-Ktx21 subfamily and therefore was classified as alpha-Ktx21.1. (C) 2011 Elsevier Ltd. All rights reserved.

Functional characterization of the Aspergillus nidulans methionine sulfoxide reductases (msrA and msrB)

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here. we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage. (C) 2009 Elsevier Inc. All rights reserved.

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirao Preto, Sao Paulo, Brazil

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeirao Preto, Sao Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All, strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors. (C) 2007 Elsevier GmbH. All rights reserved.

Evidence of caspase-mediated apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom

The aim of this work was to investigate the involvement of caspases in apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAA0. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in ""Asp and Glu"" residues. It displays high specificity toward hydrophobic L-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H(2)O(2) production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances. Published by Elsevier Inc.

Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

A proteinase, named BmooMP alpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the B beta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMP alpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMP alpha-I activity. Since the BmooMP alpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis. (C) 2007 Elsevier Ltd. All rights reserved.

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M-r = 61,000, pI similar to 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696 bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans, to improve thrombin-like activity of BjussuSP-I toxin. (c) 2007 Elsevier Masson SAS. All rights reserved.