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.

Effect of the iontophoresis of a chitosan gel on doxorubicin skin penetration and cytotoxicity

The aim of this work was to investigate doxorubicin (DOX) percutaneous absorption and retention in the skin following iontophoresis. The convective flow contribution to the overall electrotransport of DOX was also elucidated for a non-ionic hyd roxyethylcellulose gel and a cationic chitosan gel. Moreover, the cytotoxicity of DOX and its formulations, with and without low electrical current, was verified. It was observed that iontophoresis of DOX significantly increased the skin permeation and retention of the drug. In addition, the electroosmotic flow was dramatically reduced when DOX was added to the non-ionic gel, thereby indicating that the drug interacted with negative charges in the skin. Interestingly, electroosmosis was also significantly reduced when the iontophoresis was performed in the presence of the chitosan gel, but in the absence of DOX. Consequently, the transport of an electroosmotic marker from this gel almost disappeared when the positively charged drug was added to the cationic gel. These results indicated that chitosan appeared to interact with negative charges in the skin. Hence, this carrier not only reduced electroosmotic flow, but also released DOX from ionic interactions with these sites and improved its diffusion to deeper skin layers. The application of the low electrical current directly to melanoma cells increased DOX cytotoxicity by nearly three-fold, which was probably due to membrane permeation. (c) 2008 Elsevier B.V. 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.

Myotoxic phospholipases A(2) isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A(2) (PLA(2)S) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M-r similar to 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2S from snake venoms, MTX-I belonging to Asp49 PLA(2) class, enzymatically active, and MTX-II to Lys49 PLA(2)S, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA(2) and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA(2)S induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA(2) proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. (C) 2008 Elsevier Inc. All rights reserved.

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.

Antitumor effects of snake venom chemically modified Lys49 phospholipase A(2)-like BthTX-I and a synthetic peptide derived from its C-terminal region

The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115-129 C-terminal region. BPB-BthTX-1 presented cytotoxicity of 10-40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S 180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer. (C) 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Computer-aided Drug Design of Novel PLA(2) Inhibitor Candidates for Treatment of Snakebite

Phospholipases A(2) (PLA(2)) are enzymes commonly found in snake venoms from Viperidae and Elaphidae families, which are major components thereof. Many plants are used in traditional medicine its active agents against various effects induced by snakebite. This article presents the PLA(2) BthTX-I structure prediction based on homology modeling. In addition, we have performed virtual screening in a large database yielding a set of potential bioactive inhibitors. A flexible docking program was used to investigate the interactions between the receptor and the new ligands. We have performed molecular interaction fields (MIFs) calculations with the phospholipase model. Results confirm the important role of Lys49 for binding ligands and suggest three additional residues as well. We have proposed a theoretically nontoxic, drug-like, and potential novel BthTX-I inhibitor. These calculations have been used to guide the design of novel phospholipase inhibitors as potential lead compounds that may be optimized for future treatment of snakebite victims as well as other human diseases in which PLA(2) enzymes are involved.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65 kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAC-cDNA of 1548 bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58 kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Elp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. (C) 2009 Elsevier Masson SAS. All rights reserved.

Enzymatic and structural characterization of a basic phospholipase A(2) from the sea anemone Condylactis gigantea

This work aimed at the isolation and structural/functional characterization of a phospholipase A(2) (CgPLA(2)) from the extract of the anemone Condylactis gigantea. CgPLA2 was isolated with a high purity level through three chromatographic steps, showing pT8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA2 showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca2+-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA2 with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA2 revealed 84%, 61%, 43% and 42% similarity to the PLA2s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Both raps jararacussu, respectively. (C) 2010 Elsevier Masson SAS. All rights reserved.