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.

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.

Embracing ligands. A synthetic strategy towards new nitrogen-thioether multidentate ligands and characterization of the cobalt(III) complexes

The synthesis of the hexadentate ligand 2,2,9,9-tetra(methyleneamine)-4,7-dithiadecane (EtN(4)S(2)amp) is reported. The ligand is of a type in which bifurcations of the chain occur at atoms other than donor atoms. The cobalt(III) complex [Co(EtN(4)S(2)amp)](3+) (1) was isolated and characterized. The synthetic methodology also results in a number of by-products, notably 2,9,9-tris(methyleneamine)-9-methylenehydroxy-4,7-dithiadecane (Et(HO)N(3)S(2)amp) and an eleven-membered pendant arm macrocyclic ligand 6,10-dimethyl-6,10-bis(methyleneamine)-1,4-dithia-8-azaacycloundec-7- ene (dmatue). The complexes [Co(Et(HO)N(3)S(2)amp)](3+) (2), in which the alcohol is coordinated to the metal ion, and [Co(dmatue)Cl](2+) (4) were isolated and characterized. Et(HO)N(3)S(2)amp also undergoes complexation with cobalt(III) to produce two isomers endo-[Co(Et(HO) N(3)S(2)amp)Cl](2+) (endo-3) and exo-[Co(Et(HO) N(3)S(2)amp)Cl](2+) (exo-3), both with an uncoordinated alcohol group. endo- 3 has the alcohol positioned cis, and exo-3 trans, to the sixth metal coordination site. Reaction of 1 with isobutyraldehyde, paraformaldehyde and base in dimethylformamide results in the encapsulated complex [Co(1,5,5,9,13,13-hexamethyl-18,21-dithia-3,7,11,15-tetraazabicyclo[7.7.6]docosa- 3,14-diene)](ClO4)(3) . 2H(2)O ([Co(Me(6)docosadieneN(4)S(2))](3+) ( 5). All complexes have been characterized by single crystal X-ray study. The low-temperature (11 K) absorption spectrum of 1 has been measured in Nafion films with spin-allowed (1)A(1g) --> T-1(1g) and (1)A(1g) --> T-1(2g) and spin forbidden (1)A(1g) --> T-3(1g) and (1)A(1g) --> T-3(2g) bands observed. The octahedral ligand-field parameters were determined (10Dq = 22570 cm(-1), B = 551 cm(-1); C = 3500 cm(-1)). For 5 10Dq and B were determined (20580 cm(-1); 516 cm(-1), respectively) and compared with those for similar expanded cavity complexes [Co(Me(8)tricosatrieneN(6))](3+) and [Co(Me(5)tricosatrieneN(6))](3+).

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.

Increased Vascular Permeability, Angiogenesis and Wound Healing Induced by the Serum of Natural Latex of the Rubber Tree Hevea brasiliensis

Increases in vascular permeability and angiogenesis are crucial events to wound repair, tumoral growth and revascularization of tissues submitted to ischemia. An increased vascular permeability allows a variety of cytokines and growth factors to reach the damaged tissue. Nevertheless, the angiogenesis supply tissues with a wide variety of nutrients and is also important to metabolites clearance. It has been suggested that the natural latex from Hevea brasiliensis showed wound healing properties and angiogenic activity. Thus, the purpose of this work was to characterize its angiogenic activity and its effects on vascular permeability and wound healing. The serum fraction of the latex was separated from the rubber with reduction of the pH. The activity of the dialyzed serum fraction on the vascular permeability injected in subcutaneous tissue was assayed according Mile`s method. The angiogenic activity was determined using a chick embryo chorioallantoic membrane assay and its effects on the wound-healing process was determined by the rabbit ear dermal ulcer model. The serum fraction showed evident angiogenic effect and it was effective in enhancing vascular permeability. In dermal ulcers, this material significantly accelerated wound healing. Moreover, the serum fraction boiled and treated with proteases lost these activities. These results are in accordance with the enhancement of wound healing observed in clinical trials carried out with a biomembrane prepared with the same natural latex. Copyright (C) 2009 John Wiley & Sons, Ltd.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

Network structures of Bis-GMA/TEGDMA resins differ in DC, shrinkage-strain, hardness and optical properties as a function of reducing agent

Objectives. To evaluate the influence of different tertiary amines on degree of conversion (DC), shrinkage-strain, shrinkage-strain rate, Knoop microhardness, and color and transmittance stabilities of experimental resins containing BisGMA/TEGDMA (3: 1 wt), 0.25wt% camphorquinone, 1wt% amine (DMAEMA, CEMA, DMPT, DEPT or DABE). Different light-curing protocols were also evaluated. Methods. DC was evaluated with FTIR-ATR and shrinkage-strain with the bonded-disk method. Shrinkage-strain-rate data were obtained from numerical differentiation of shrinkage-strain data with respect to time. Color stability and transmittance were evaluated after different periods of artificial aging, according to ISO 7491: 2000. Results were evaluated with ANOVA, Tukey, and Dunnett`s T3 tests (alpha = 0.05). Results. Studied properties were influenced by amines. DC and shrinkage-strain were maximum at the sequence: CQ < DEPT < DMPT <= CEMA approximate to DABE < DMAEMA. Both DC and shrinkage were also influenced by the curing protocol, with positive correlations between DC and shrinkage-strain and DC and shrinkage-strain rate. Materials generally decreased in L* and increased in b*. The strong exception was the resin containing DMAEMA that did not show dark and yellow shifts. Color varied in the sequence: DMAEMA < DEPT < DMPT < CEMA < DABE. Transmittance varied in the sequence: DEPT approximate to DABE < DABE approximate to DMPT approximate to CEMA < DMPT approximate to CEMA approximate to DMAEMA, being more evident at the wavelength of 400 nm. No correlations between DC and optical properties were observed. Significance. The resin containing DMAEMA showed higher DC, shrinkage-strain, shrinkage-strain rate, and microhardness, in addition to better optical properties. (C) 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Effect of repeated cycles of chemical disinfection on the roughness and hardness of hard reline acrylic resins

Objective: The aim of this study was to assess the effect of repeated cycles of five chemical disinfectant solutions on the roughness and hardness of three hard chairside reliners. Methods: A total of 180 circular specimens (30 mm x 6 mm) were fabricated using three hard chairside reliners (Jet; n = 60, Kooliner; n = 60, Tokuyama Rebase II Fast; n = 60), which were immersed in deionised water (control), and five disinfectant solutions (1%, 2%, 5.25% sodium hypochlorite; 2% glutaraldehyde; 4% chlorhexidine gluconate). They were tested for Knoop hardness (KHN) and surface roughness (mu m), before and after 30 simulated disinfecting cycles. Data was analysed by the factorial scheme (6 x 2), two-way analysis of variance (anova), followed by Tukey`s test. Results: For Jet (from 18.74 to 13.86 KHN), Kooliner (from 14.09 to 8.72 KHN), Tokuyama (from 12.57 to 8.28 KHN) a significant decrease in hardness was observed irrespective of the solution used on all materials. For Jet (from 0.09 to 0.11 mu m) there was a statistically significant increase in roughness. Kooliner (from 0.36 to 0.26 mu m) presented a statistically significant decrease in roughness and Tokuyama (from 0.15 to 0.11 mu m) presented no statistically significant difference after 30 days. Conclusions: This study showed that all disinfectant solutions promoted a statistically significant decrease in hardness, whereas with roughness, the materials tested showed a statistically significant increase, except for Tokuyama. Although statistically significant values were registered, these results could not be considered clinically significant.

Towards synthetic vaccines built on carbohydrate cores

Lipophilic polyfunctional carbohydrate core/templates have been designed and developed for drug/vaccine delivery. Three carbohydrate-based templates containing four protected N-terminal arms were synthesised from glucose and galactose. Methyl alpha-D-glucopyranoside was converted to two derivatives bearing a carboxylic acid handle for attachment to solid supports, spacer arms of differing hydrophilicity, and phthaloyl-protected amino groups suitable for peptide chain extension. beta-D-Galactopyranosyl azide was converted to a template bearing a carboxylic acid handle and four BOC-protected amines. All the templates were found to be suitable for attachment to solid supports and subsequent cleavage from resins, using either BOC- or FMOC-methodologies.

The effects of fillers on the chemorheology of highly filled epoxy resins: I. Effects on cure transitions and kinetics

This work examines the effects of level of silica filler (at 0, 10, 30, 50wt%) on the gelation and vitrification of a model silica-filled diglycidyl ether of bisphenol F (DGEBF)/methylenedianiline (MDA) system. An increased filler level is shown to decrease the gelation and vitrification times at low temperatures (below 80degreesC). FTIR cure kinetics show that the reaction rates are increased and the activation energies of gelation are reduced at these temperatures, indicating that network formation is made easier. Entropic and catalytic reasons for this phenomenon are discussed. (C) 2003 Society of Chemical Industry.

The Relative Frequency of the Synthetic End Composite Futures in the Newspaper Quest-France and some Observations on Distribution

Figures on the relative frequency of synthetic and composite future forms in Ouest-France are presented and compared with those of earlier studies on the passé simple and passé composé. The synthetic future is found to be dominant. Possible formal explanations for distribution are found to be inconclusive. Distribution across different text-types is found to be more promising, since contrastive functions of the two forms can be identified in texts where they co-occur. The composite future typically reports new proposals or plans as current news, while the synthetic future outlines details that will be realised at the time of implementation. Both functions are important in dailies, but current news is more often expressed in the present tense at the expense of the composite future.

Evaluation of organophosphorus and synthetic pyrethroid insecticides against six vector mosquitoe species

Three organophosphorus compounds- malathion, folithion and temephos- and two synthetic pyrethroids- alphamethrin and deltamethrin- were used for monitoring the susceptibility status of larvae and adults of six vector mosquitoe species: Culex quinquefasciatus (Filariasis) and Aedes albopictus (Dengue) (both laboratory and field strains); laboratory strains of Aedes aegypti (Dengue), Anopheles slephensi and Anopheles culicifacies (Malaria), and Culex tritaeniorhynchus (Japanese encephalitis) in India. From the LC50 values obtained for these insecticides, it was found that all mosquito species including the field strains of Cx. quinquefasciatus and Ae. albopictus were highly susceptible Except for Cx. quinquefasciatus (field strain) against malathion, 100% mortality was observed at the discriminating dosages recommended by World Health Organization. The residual effect of alphamethrin, deltamethrin, malathion and folithion at 25 mg (ai)/m² on different surfaces against six species of vector mosquitoes showed that alphamethrin was the most effective on all four treated surfaces (mud, plywood, cement and thatch). Nevertheless, residual efficacy lasted longer on thatch than on the other surfaces. Therefore, synthetic pyrethroids such as alphamethrin can be effectively employed in integrated vector control operations.

Design of a new test chamber for evaluation of the toxicity of rubber infill

A test chamber was projected and built (according to ISO 16000-9 Standard) to simulate atmospheric conditions experienced by rubber infill (when applied in synthetic turf pitches) and measure accurately the airborne emissions of pollutants such as dusts and volatile organic compounds (VOC), as well as pollutants present in leachates. It should be pointed out that standard ISO 16000-9 is only concerned with the determination of the emission of VOC from building products and furnishing (not specific of synthetic turf materials), whereas other standards are concerned with the emission of leachates only. This procedure is to be considered as a technical option to the lysimeter "global turf system evaluation" when the rubber infill alone is to be evaluated. The advantage of the proposed option considering this "test chamber" is its simplicity and economy. This test chamber is actually installed and being used for tests in LAIST.