Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM`s inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 mu M Ca(2+), DHM (50-250 mu M) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 mu M) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline. (C) 2009 Elsevier Ltd. All rights reserved.

Ca(2+) binding to c-state of adenine nucleotide translocase (ANT)-surrounding cardiolipins enhances (ANT)-Cys(56) relative mobility: A computational-based mitochondrial permeability transition study

The oxidation of critical cysteines/related thiols of adenine nucleotide translocase (ANT) is believed to be an important event of the Ca(2+)-induced mitochondrial permeability transition (MPT), a process mediated by a cyclosporine A/ADP-sensitive permeability transition pores (PTP) opening. We addressed the ANT-Cys(56) relative mobility status resulting from the interaction of ANT/surrounding cardiolipins with Ca(2+) and/or ADP by means of computational chemistry analysis (Molecular Interaction Fields and Molecular Dynamics studies), supported by classic mitochondrial swelling assays. The following events were predicted: (i) Ca(2+) interacts preferentially with the ANT surrounding cardiolipins bound to the H4 helix of translocase, (ii) weakens the cardiolipins/ANT interactions and (iii) destabilizes the initial ANT-Cys(56) residue increasing its relative mobility. The binding of ADP that stabilizes the conformation ""m"" of ANT and/or cardiolipin, respectively to H5 and H4 helices, could stabilize their contacts with the short helix h56 that includes Cys(56), accounting for reducing its relative mobility. The results suggest that Ca(2+) binding to adenine nucleotide translocase (ANT)-surrounding cardiolipins in c-state of the translocase enhances (ANT)-Cys(56) relative mobility and that this may constitute a potential critical step of Ca(2+)-induced PTP opening. (C) 2009 Elsevier B.V. All rights reserved.

Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell

Paracoccidioides brasiliensis infectious process relies on the initial expression of virulence faactors that are assumed to be controlled by molecular mechanisms through which the conidia and/or mycelial fragments convert to yeast cells. In order to analyze the profile of the thermally-induced dimorphic gene expression, 48 h C-L transition cultures which had been incubated at 36 degrees C were studied. By this time approximately 50% of the conidial population had already reverted to yeast form cells. At this transition time, an EST-Orestes library was constructed and characterized. As a result, 79 sequences were obtained, of which 39 (49.4%) had not been described previously in other libraries of this fungus and which could represent novel exclusive C-Y transition genes. Two of these sequences are, among others, cholestanol delta-isomerase, and electron transfer flavoprotein-ubiquinoneoxidoreductase (ETF-QO). The other 40 (50.6%) sequences were shared with Mycelia (M), Yeast (Y) or Mycelia to yest transition (M-Y) libraries. An important component of this group of sequences is a putative response regulator receiver SKN7, a protein of high importance in stress adaptation and a regulator of virulence in some bacteria and fungi. This is the first report identifying genes expressed during the C-Y transition process, the initial step required to understand the natural history of P brasiliensis conidia induced infection.

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.

Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom

The present article describes an L-amino acid oxidase from Bothrops atrox snake venom as with antiprotozoal activities in Trypanosoma cruzi and in different species of Leishmania (Leishmania braziliensis, Leishmania donovani and Leishmania major). Leishmanicidal effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Leishmania spp. cause a spectrum of diseases, ranging from self-healing ulcers to disseminated and often fatal infections, depending on the species involved and the host`s immune response. BatroxLAAO also displays bactericidal activity against both Gram-positive and Gram-negative bacteria. The apoptosis induced by BatroxLAAO on HL-60 cell lines and PBMC cells was determined by morphological cell evaluation using a mix of fluorescent dyes. As revealed by flow cytometry analysis, suppression of cell proliferation with BatroxLAAO was accompanied by the significant accumulation of cells in the G0/G1 phase boundary in HL-60 cells. BatroxLAAO at 25 mu g/mL and 50 mu g/mL blocked G0-G1 transition, resulting in G0/G1 phase cell cycle arrest, thereby delaying the progression of cells through S and G2/M phase in HL-60 cells. This was shown by an accentuated decrease in the proportion of cells in S phase, and the almost absence of G2/M phase cell population. BatroxLAAO is an interesting enzyme that provides a better understanding of the ophidian envenomation mechanism, and has biotechnological potential as a model for therapeutic agents. (C) 2011 Elsevier Masson SAS. All rights reserved.

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.

Analysis of the Phase Changes During Evaporation of Emulsions with Different Oil Phases

Emulsions surfer alterations in their microstructure after applied on the skin, because of the interaction with skin constituents and mainly by the evaporation of volatile components. These alterations are not even considered by cosmetic formulators, but they are extremely important because they can act on formulation stability, on delivery and on permeation of actives and also on the ability to build the occlusive film, responsible for skin`s moisturization. This research studied the phase changing during evaporation of emulsions made with three different oil phase: mineral oil, avocado oil, and isocethyl/stearoil stearate, as a function of the decrease on water ratio, using phase diagrams and evaporation test. It was observed the formation of liquid crystalline phases and their transition along the evaporation path for emulsions with the three different oil phases. It was also observed that these transitions occurred in different water ratios.

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

Entanglement in a simple quantum phase transition

What entanglement is present in naturally occurring physical systems at thermal equilibrium? Most such systems are intractable and it is desirable to study simple but realistic systems that can be solved. An example of such a system is the one-dimensional infinite-lattice anisotropic XY model. This model is exactly solvable using the Jordan-Wigner transform, and it is possible to calculate the two-site reduced density matrix for all pairs of sites. Using the two-site density matrix, the entanglement of formation between any two sites is calculated for all parameter values and temperatures. We also study the entanglement in the transverse Ising model, a special case of the XY model, which exhibits a quantum phase transition. It is found that the next-nearest-neighbor entanglement (though not the nearest-neighbor entanglement) is a maximum at the critical point. Furthermore, we show that the critical point in the transverse Ising model corresponds to a transition in the behavior of the entanglement between a single site and the remainder of the lattice.

Magnetic susceptibility of the normal-superconducting transition in high-purity single-crystal α-uranium

We report complex ac magnetic susceptibility measurements of a superconducting transition in very high-quality single-crystal alpha-uranium using microfabricated coplanar magnetometers. We identify an onset of superconductivity at Tapproximate to0.7 K in both the real and imaginary components of the susceptibility which is confirmed by resistivity data. A superconducting volume fraction argument, based on a comparison with a calibration YBa2Cu3O7-delta sample, indicates that superconductivity in these samples may be filamentary. Our data also demonstrate the sensitivity of the coplanar micro-magnetometers, which are ideally suited to measurements in pulsed magnetic fields exceeding 100 T.

Half-Filled Layered Organic Superconductors and the Resonating-Valence-Bond Theory of the Hubbard-Heisenberg Model

We present a resonating-valence-bond theory of superconductivity for the Hubbard-Heisenberg model on an anisotropic triangular lattice. Our calculations are consistent with the observed phase diagram of the half-filled layered organic superconductors, such as the beta, beta('), kappa, and lambda phases of (BEDT-TTF)(2)X [bis(ethylenedithio)tetrathiafulvalene] and (BETS)(2)X [bis(ethylenedithio)tetraselenafulvalene]. We find a first order transition from a Mott insulator to a d(x)(2)-y(2) superconductor with a small superfluid stiffness and a pseudogap with d(x)(2)-y(2) symmetry.