Isolation, characterization and biological activity of acidic phospholipase A(2) isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIIB diffracted beyond 1.8 Angstrom resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 Angstrom, b = 54.2 Angstrom and c = 90.7 Angstrom. The crystal structure has been refined to a crystallographic residual of 16.1% (R-free = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Active planar waveguides based on sol-gel Er3+-doped SiO2-ZrO2 for photonic applications: Morphological, structural and optical properties

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier B.V. All rights reserved.

Comparative study using small-angle x-ray scattering and nitrogen adsorption in the characterization of silica xerogels and aerogels

A comparative study using small-angle x-ray scattering (SAXS) and nitrogen adsorption has been carried out in the structural characterization of silica xerogels and aerogels, obtained from tetraethoxysilane sonohydrolysis. The specific surface and the mean pore size as measured by both the techniques were found to be in notable agreement in all cases for aerogels and xerogels. According to the SAXS data, aerogels at 500 °C exhibit a mass fractal structure with fractal dimension D∼2.4 in the range between the correlation length ξ∼5.3 nm and a∼0.75 nm. An experimental method to probe the mass fractal structure of aerogels from exclusively nitrogen adsorption isotherms has been presented. For aerogels at 500 °C, we have found D∼2.4 in the range between the pore width 2rξ∼33 nm and 2ra∼4.5 nm, which is in notable agreement with the SAXS results (D ∼2.4, ξ∼5.3 nm, a∼0.75 nm) if we assign the pore width 2r probed by the Kelvin equation in the adsorption method to the Bragg distance 2π/q associated to the correlation length 1/q probed by SAXS.

Microstructural characterization of layers produced by plasma nitriding on austenitic and superaustenitic stainless steel grades

High chromium content is responsible for the formation of a protective passive surface layer on austenitic stainless steels (ASS). Due to their larger amounts of chromium, superaustenitic stainless steels (SASS) can be chosen for applications with higher corrosion resistance requirements. However, both of them present low hardness and wear resistance that has limited their use for mechanical parts fabrication. Plasma nitriding is a very effective surface treatment for producing harder and wear resistant surface layers on these steel grades, without harming their corrosion resistance if low processing temperatures are employed. In this work UNS S31600 and UNS S31254 SASS samples were plasma nitrided in temperatures from 400 °C to 500 °C for 5 h with 80% H 2-20% N2 atmosphere at 600Pa. Nitrided layers were analyzed by optical (OM) and transmission electron microscopy (TEM), x-ray diffraction (XRD), and Vickers microhardness testing. Observations made by optical microscopy showed that N-rich layers were uniform but their thicknesses increased with higher nitriding temperatures. XRD analyses showed that lower temperature layers are mainly composed by expanded austenite, a metastable nitrogen supersaturated phase with excellent corrosion and tribological properties. Samples nitrided at 400 °C produced a 5 μm thick expanded austenite layer. The nitrided layer reached 25 lm in specimens treated at 500 °C. There are indications that other phases are formed during higher temperature nitriding but XRD analysis was not able to determine that phases are iron and/or chromium nitrides, which are responsible for increasing hardness from 850 up to 1100 HV. In fact, observations made by TEM have indicated that formation of fine nitrides, virtually not identified by XRD technique, can begin at lower temperatures and their growth is affected by both thermodynamical and kinetics reasons. Copyright © 2012 by ASTM International.

Study of a series of cobalt(II) sulfonamide complexes: Synthesis, spectroscopic characterization, and microbiological evaluation against M. tuberculosis. Crystal structure of [Co(sulfamethoxazole)2(H 2O)2]·H2O

Nowadays, the research for new and better antimicrobial compounds is an important field due to the increase of immunocompromised patients, the use of invasive medical procedures and extensive surgeries, among others, that can affect the incidence of infections. Another big problem associated is the occurrence of drug-resistant microbial strains that impels a ceaseless search for new antimicrobial agents. In this context, a series of heterocyclic- sulfonamide complexes with Co(II) was synthesized and characterized with the aim of obtaining new antimicrobial compounds. The structural characterization was performed using different spectroscopic methods (UV-Vis, IR, and EPR). In spite of the fact that the general stoichiometry for all the complexes was Co(sulfonamide)2·nH2O, the coordination atoms were different depending on the coordinated sulfonamide. The crystal structure of [Co(sulfamethoxazole)2(H2O)2]·H 2O was obtained by X-ray diffraction showing that Co(II) is in a slightly tetragonal distorted octahedron where sulfamethoxazole molecules act as a head-to-tail bridges between two cobalt atoms, forming polymeric chains. Besides, the activity against Mycobacterium tuberculosis, one of the responsible for tuberculosis, and the cytotoxicity on J774A.1 macrophage cells were evaluated. © 2012 Elsevier B.V. All rights reserved.

Synthesis and characterization of melanin in DMSO

Recently soluble melanin derivatives have been obtained by a synthetic procedure carried out in DMSO (D-melanin). In this work a comparative study of the structural characteristics of synthetic melanin derivatives obtained by oxidation of L-DOPA in H2O and DMSO are presented. To this end, Fourier-transform infrared spectroscopy as well as proton and carbon nuclear magnetic resonance techniques has been employed. In addition, aging effects have been investigated for D-melanin. The results suggest that sulfonate groups (-SO2CH3) from the oxidation of DMSO, are incorporated into melanin, which confers protection to the phenolic hydroxyl group present in its structure. The solubility of D-melanin in DMSO is attributed to the presence of these groups. When D-melanin is left in air for long time periods, the sulfonate groups leave the structure, and an insoluble compound is obtained. NaOH and water have been used, in order to accelerate the release of the sulfonate groups attached to D-melanin, thereby corroborating the proposed structure and the synthesis mechanism. © 2013.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)

Biochemical and enzymatic characterization of BpMP-I, a fibrinogenolytic metalloproteinase isolated from Bothropoides pauloensis snake venom

Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SOS-PAGE and showed a single chain polypeptide of 20 kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and beta-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme. (C) 2011 Elsevier Inc. All rights reserved.

Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom

A fibrinogenolytic metalloproteinase from Bothrops moojeni venom, named moojenin, was purified by a combination of ion-exchange chromatography on DEAE-Sephacel and gel filtration on Sephacryl S-300. SDS-PAGE analysis indicated that moojenin consists of a single polypeptide chain and has a molecular mass about 45 kDa. Sequencing of moojenin by Edman degradation revealed the amino acid sequence LGPDIVSPPVCGNELLEV-GEECDCGTPENCQNE, which showed strong identity with many other snake venom metalloproteinases (SVMPs). The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the E beta-chain, and shows no effects on the gamma-chain. Moojenin showed a coagulant activity on bovine plasma about 3.1 fold lower than crude venom. The fibrinogenolytic and coagulant activities of the moojenin were abolished by preincubation with EDTA, 1,10-phenanthroline and beta-mercaptoethanol. Moojenin showed maximum activity at temperatures ranging from 30 to 40 degrees C and its optimal pH was 4.0. Its activity was completely lost at temperatures above 50 degrees C. Moojenin induced necrosis in liver and muscle, evidenced by morphological alterations, but did not cause histological alterations in mouse lungs, kidney or heart. Moojenin rendered the blood uncoagulatable when it was intraperitoneally administered into mice. This metalloproteinase may be of medical interest because of its anticoagulant activity. (C) 2012 Elsevier Ltd. All rights reserved.

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the B beta chain and BpirSP41 on both A alpha and B beta chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of similar to 3.5 mu g versus 20 mu g for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu2+ ion and specific serine protease inhibitors. In addition. BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure-function relationship of SVSPs. (C) 2012 Elsevier Masson SAS. All rights reserved.

Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.

Characterization of the environment /

"Publication no. FHWA-RD-00-030."

Structural Insights into Viral Membrane Fusion Machinery via Cryo-Electron Tomography: Influenza Virus and Human Parainfluenza Virus

Thesis (Ph.D.)--University of Washington, 2016-06

Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.