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.

Acute Toxicity of Schizolobium parahyba Aqueous Extract in Mice

The herbal extract of Schizolobium parahyba leaves is used commonly in the Brazil central region to treat snakebites. This study evaluates the acute toxicological effects of Schizolobium parahyba aqueous extract in mice 24 h after intraperitoneal administration. Acute toxicity was evaluated using biochemical, hematological and histopathological assays. Alterations in the levels of transaminases, bilirubin, albumin and prothrombrin time were observed, and these are likely to occur due to hepatic injury, which was confirmed by light microscopy. Liver histopathological analysis revealed the presence of lymph plasmocitary inflammatory infiltrate, but no other histopathological alterations were observed in any of the other organs analysed. The data confirm the low toxicity of the extract of Schizolobium parahyba and provide a model for the selection of a dose that does not cause injuries in the organism. Copyright (C) 2009 John Wiley & Sons, Ltd.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M(r) = 34,000 under reducing conditions and pI similar to 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (-70 to 37 degrees C), pH values (3-9) or in the presence of divalent metal ions (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom. (C) 2009 Elsevier Ltd. All rights reserved.

Expression of Human Recombinant Antibody Fragments Capable of Partially Inhibiting the Phospholypase Activity of Crotalus durissus terrificus Venom

Crotoxin is the main toxic component of the South American rattlesnake Crotalus durissus terrificus venom. It is composed of two different subunits: CA, crotapotin, and CB (basic subunit of cortoxin isolated from C. d. terrificus), a weakly toxic phospholipase A(2) with high enzymatic activity. The phospholipases A(2) are abundant in snake venoms and are responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. However, in addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxic, myotoxic, oedema-inducing, hypotensive, platelet-aggregating, cardiotoxic, and anticoagulant effects have been attributed to venom phospholipases A(2). In this study, we used a non-immune human single-chain fragment variable library, Griffin.1 (Medical Research Council, Cambridge, UK) for selection of recombinant antibodies against antigens present in C. d. terrificus venom and identification of specific antibodies able to inhibit the phospholipase activity. Two clones were identified as capable of inhibiting partially this activity in vitro. These clones were able to reduce in vivo the myotoxic and oedema-inducing activity of CB and the lethality of C. d. terrificus venom and crotoxin, but had no effect on the in vitro anticoagulant activity of CB. These results demonstrate the potential of using recombinant single-chain fragment variable libraries in the production of antivenoms.

Bioactive Chemical Constituents and Comparative Antimicrobial Activity of Callus Culture and Adult Plant Extracts from Alternanthera tenella

Crude extracts of a callus culture (two culture media) and adult plants (two collections) from Alternanthera tenella Colla (Amaranthaceae) were evaluated for their antibacterial and antifungal activity, in order to investigate the maintenance of antimicrobial activity of the extracts obtained from plants in vivo and in vitro. The antibacterial and antifungal activity was determined against thirty strains of microorganisms including Gram-positive and Gram-negative bacteria, yeasts and dermatophytes. Ethanolic and hexanic extracts of adult plants collected during the same period of the years 1997 and 2002 [Ribeirao Preto (SP), collections 1 and 2] and obtained from plant cell callus culture in two different hormonal media (AtT43 and AtT11) inhibited the growth of bacteria, yeasts and dermatophytes with inhibition halos between 6 and 20 mm. For the crude extracts of adult plants bioassay-guided fractionation, purification, and isolation were performed by chromatographic methods, and the structures of the isolated compounds were established by analysis of chemical and spectral evidences (UV, IR, NMR and ES-MS). Steroids, saponins and flavonoids (aglycones and C-glycosides) were isolated. The minimum inhibitory concentration (MIC) of the isolated compounds varied from 50 to 500 mu g/mL.

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.

Lipase Production by Endophytic Fungus Cercospora kikuchii: Stability of Enzymatic Activity after Spray Drying in the Presence of Carbohydrates

The present work deals with improving the production and stabilization of lipases from Cercospora kikuchii. Maximum enzyme production (9.384 U/ml) was obtained after 6 days in a medium supplemented with 2% soybean oil. The lipases were spray dried with different adjuvants, and their stability was studied. The residual enzyme activity after drying with 10% (w/v) of lactose, b- cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63 to 100%. The enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5 degrees C and 40% at 25 degrees C after 8 months. The lipase dried with 10% of beta-cyclodextrin retained 72% of activity at 5 degrees C. Lipases were separated by butyl-sepharose column into 4 pools, and pool 4 was partially purified (33.1%; 269.5 U/mg protein). This pool was also spray dried in maltodextrin DE10, and it maintained 100% of activity.

Ultramicroporous membranes for hydrogen separation

Fuel cell systems offer excellent efficiencies when compared to internal combustion engines, which result in reduced fuel consumption and greenhouse gas emissions. One of the areas requiring research for the success of fuel cell technology is the H2 fuel purification to reduce CO, which is a poison to fuel cells. Molecular sieve silica (MSS) membranes have a potential application in this area. In this work showed activated transport, a characteristic of ultramicroporous (dp

High selectivity microporous silica membranes for lactic acid dehydration

Lactic acid (LA) has significant market potential for many industries including food, cosmetics, pharmaceuticals, medical and biodegradable materials. Production of LA usually begins with the fermentation of glucose but subsequent stages for the enrichment of lactic acid are complex and energy intensive and could be minimised using water selective membrane technology. In this work, we trialled a highly selective hydrostable carbonised template molecular sieve silica (CTMSS) membrane for the dehydration of a 15 vol% aqueous lactic acid solution with 0.1 vol% glucose. CTMSS membrane films were developed by dip-coating ceramic substrates with silica sols made using the acid catalysed sol-gel process. Permeation was performed by feeding LA/glucose solution to the membrane cell at 18°C in a standard pervaporation setup. The membrane showed selective transport of water from the aqueous feed to the permeate while glucose was not detected. CTMSS membrane permeate flux stabilised at 0.2 kg.m-2.hr-1 in 3.9 hours, and reduced LA to lower than 0.2 vol%. Flux through the CTMSS micropores was activated, displaying increased initial flux to 1.58 kg.m-2.hr-1 at 60°C. To enrich a 1 l.min-1 stream to 85% LA in a single stage, a minimum membrane area of 324 m2 would be required at 18°C. Increased operating temperature to 80°C significantly reduced this area to 24 m2 but LA levels in the permeate stream increased to 0.5 vol%. The highly selective CTMSS membrane technology is an ideal candidate for LA purification. CTMSS membrane systems operate stably in aqueous systems leading to potential cost reductions in LA processing for future markets.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca

Albicidins are a family of phytotoxins and antibiotics which play an important role in the pathogenesis of sugarcane leaf scald disease. The albA gene from Klebsiella oxytoca encodes a protein which inactivates albicidin by heat-reversible binding. Albicidin ligand binding to a recombinant AlbA protein, purified by means of a glutathione S-transferase gene fusion system, is an almost instant and saturable reaction. Kinetic and stoichiometric analysis of the binding reaction indicated the presence of a single high affinity binding site with a dissociation constant of 6.4 x 10(-8) M. The AlbA-albicidin complex is stable from 4 to 40 degrees C, from ph 5 to 9 and in high salt solutions. Treatment with protein denaturants released all bound albicidin. These properties indicate that AlbA may be a useful affinity matrix for selective purification of albicidin antibiotics. AlbA does not bind to p-nitrophenyl butyrate or alpha-naphthyl butyrate, the substrates of the albicidin detoxification enzyme AlbD from Pantoea dispersa. The potential exists to pyramid genes for different mechanisms in transgenic plants to protect plastid DNA replication from inhibition by albicidins.

Bacterial expression of two human aryl sulfotransferases

The effect of replacing a single codon in the N-terminal of human aryl sulfotransferase (HAST) 1 and 3 with one that is more commonly found in E. coli genes was assessed. The pKK233-2 E. coli expression vector was employed and the polymerase chain reaction (PCR) was used to introduce the 5' nucleotide substitution, at the same time maintaining the fidelity of the amino acid sequence. The data indicates that this change had a minimal effect on protein production, subcellular localization or, in the case of HAST3, catalytic activity. In general, the pKK233-2 E. coli vector has been less than optimal for expressing human sulfotransferase cDNAs. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

A protein with protective properties against the cellular defense reactions in insects

The molecular mechanism of how insects recognize intruding microorganisms and parasites and distinguish them from own body structures is not well known. We explored evolutionary adaptations in an insect parasitoid host interaction to identify components that interfere with the recognition of foreign objects and cellular encapsulation. Because some parasitoids provide protection for the developing wasp in the absence of an overt suppression of the insect host defense, we analyzed the surface of eggs and symbiotic viruses for protective properties. Here we report on the molecular cloning of a 32-kDa protein (Crp32) that is one of the major protective components. It is produced in the calyx cells of the female wasp ovaries and attached to the surface of the egg and other particles including polydnaviruses. The recombinant protein confers protection to coated objects in a cellular encapsulation assay suggesting that a layer of Crp32 may prevent cellular encapsulation reactions by a local inactivation of the host defense system.

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1-3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modifications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4-6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4-6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man. These glycans are linked together by the conserved phosphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Man alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

Hydromorphone-3-glucuronide: Biochemical synthesis and preliminary pharmacological evaluation

Hydromorphone-3-glucuronide (H3G) was synthesized biochemically using rat liver microsomes, uridine-5'-diphosphoglucuronic acid (UDPGA) and the substrate, hydromorphone. Initially, the crude putative H3G product was purified by ethyl acetate precipitation and washing with acetonitrile, Final purification was achieved using semi-preparative high-performance-liquid-chromatography (HPLC) with ultraviolet (UV) detection. The purity of the final H3G product was shown by HPLC with electrochemical and ultraviolet detection to be > 99.9% and it was produced in a yield of approximate to 60% (on a molar basis). The chemical structure of the putative H3G was confirmed by enzymatic hydrolysis of the glucuronide moiety using P-glucuronidase, producing a hydrolysis product with the same HPLC retention time as the hydromorphone reference standard. Using HPLC with tandem mass spectrometry (HPLC-MS-MS) in the positive ionization mode, the molecular mass (M+1) was found to be 462 g/mol, in agreement with H3G's expected molecular weight of 461 g/mol. Importantly, proton-NMR indicated that the glucuronide moiety was attached at the 3-phenolic position of hydromorphone. A preliminary evaluation of H3G's intrinsic pharmacological effects revealed that following icy administration to adult male Sprague-Dawley rats in a dose of 5 mu g, H3G evoked a range of excitatory behavioural effects.including chewing, rearing, myoclonus, ataxia and tonic-clonic convulsions, in a manner similar to that reported previously for the glucuronide metabolites of morphine, morphine-3-glucuronide and normorphine-3-glucuronide.