Impedimetric immunosensor for electronegative low density lipoprotein (LDL(-)) based on monoclonal antibody adsorbed on (polyvinyl formal)-gold nanoparticles matrix

Monoclonal antibodies (MAb) have been commonly applied to measure LDL in vivo and to characterize modifications of the lipids and apoprotein of the LDL particles. The electronegative low density lipoprotein (LDL(-)) has an apolipoprotein B-100 modified at oxidized events in vivo. In this work, a novel LDL-electrochemical biosensor was developed by adsorption of anti-LDL(-) MAb on an (polyvinyl formal)-gold nanoparticles (PVF-AuNPs)-modified gold electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the recognition of LDL-. The interaction between MAb-LDL(-) leads to a blockage in the electron transfer of the [Fe(CN)(6)](4-)/K(4)[Fe(CN)(6)](3-) redox couple, which may could result in high change in the electron transfer resistance (R(CT)) and decrease in the amperometric responses in CV analysis. The compact antibody-antigen complex introduces the insulating layer on the assembled surface, which increases the diameter of the semicircle, resulting in a high R(CT), and the charge transferring rate constant k(0) decreases from 18.2 x 10(-6) m/s to 4.6 x 10(-6) m/s. Our results suggest that the interaction between MAb and lipoprotein can be quantitatively assessed by the modified electrode. The PVF-AuNPs-MAb system exhibited a sensitive response to LDL(-), which could be used as a biosensor to quantify plasmatic levels of LDL(-). (C) 2011 Elsevier B.V. All rights reserved.

Permeation of Large Antineoplastics into Wild Strain of Saccharomyces cerevisiae

Saccharomyces cerevisiae has been used in genotoxicity and cytotoxicity assays for several years before the Ames Test approach. However the cell permeability of yeast has been considered a limitant factor to this kind of assay and many researchers have been introducing genetic modifications into wild strains to improve the sensitivity to chemical compounds. In our study, we used Saccharomyces cerevisiae ATCC 9763, well known and very common strain in antibiotic assays, and we evaluated the cytotoxicity of some antineoplastic agents (etoposide, epirubicin, carboplatin, cisplatin and mitoxantrone). Each culture was observed under the light of microscope and photographed. Neither genetic modification nor addition of permeation inducers, as dimethylsulfoxide (DMSO), were introduced during the assays and the cells presented good sensitivity to those compounds, demonstrating that other potential strains and characteristics of cells should be reconsidered to improve these assays apart from the cellular permeability.

Prerequisite Programs at Schools: Diagnosis and Economic Evaluation

In this study, 20 Brazilian public schools have been assessed regarding good manufacturing practices and standard sanitation operating procedures implementation. We used a checklist comprised of 10 parts ( facilities and installations, water supply, equipments and tools, pest control, waste management, personal hygiene, sanitation, storage, documentation, and training), making a total of 69 questions. The implementing modification cost to the found nonconformities was also determined so that it could work with technical data as a based decision-making prioritization. The average nonconformity percentage at schools concerning to prerequisite program was 36%, from which 66% of them own inadequate installations, 65% waste management, 44% regarding documentation, and 35% water supply and sanitation. The initial estimated cost for changing has been U.S.$24,438 and monthly investments of 1.55% on the currently needed invested values. This would result in U.S.$0.015 increase on each served meal cost over the investment replacement within a year. Thus, we have concluded that such modifications are economically feasible and will be considered on technical requirements when prerequisite program implementation priorities are established.

Brazilian flavonoid database: Application of quality evaluation system

Much information on flavonoid content of Brazilian foods has already been obtained; however, this information is spread in scientific publications and non-published data. The objectives of this work were to compile and evaluate the quality of national flavonoid data according to the United States Department of Agriculture`s Data Quality Evaluation System (USDA-DQES) with few modifications, for future dissemination in the TBCA-USP (Brazilian Food Composition Database). For the compilation, the most abundant compounds in the flavonoid subclasses were considered (flavonols, flavones, isoflavones, flavanones, flavan-3-ols, and anthocyanidins) and the analysis of the compounds by HPLC was adopted as criteria for data inclusion. The evaluation system considers five categories, and the maximum score assigned to each category is 20. For each data, a confidence code (CC) was attributed (A, B, C and D), indicating the quality and reliability of the information. Flavonoid data (773) present in 197 Brazilian foods were evaluated. The CC ""C"" (as average) was attributed to 99% of the data and ""B"" (above average) to 1%. The main categories assigned low average scores were: number of samples; sampling plan and analytical quality control (average scores 2, 5 and 4, respectively). The analytical method category received an average score of 9. The category assigned the highest score was the sample handling (20 average). These results show that researchers need to be conscious about the importance of the number and plan of evaluated samples and the complete description and documentation of all the processes of methodology execution and analytical quality control. (C) 2010 Elsevier Inc. All rights reserved.

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex - A system capable of producing nitric oxide and singlet oxygen

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc = phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO) (ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Nitric oxide photorelease from hydrogels and from skin containing a nitro-ruthenium complex

Nitric oxide (NO) is a gaseous molecule that has specific functions dictated by its localization and its kinetics of release. As NO-donors have a range of potential uses in the skin, much attention has been paid to the development of topical NO delivery systems. The aim of this work was to study the release rate and the skin penetration of the NO-donor cis[Ru(NO(2))(bpy)(2)(4-pic)](+) from different gel formulations and their potential as topical NO delivery systems under light stimuli. Among the formulations developed, the anionic gel retarded the nitro-ruthenium complex diffusion and also obstructed NO release after light irradiation. On the other hand, NO release before light irradiation was observed when the complex was dispersed in the cationic chitosan gel, possibly due to oxi-redox reactions between the amino groups of the polymer and the drug molecule. Finally, the non-ionic gel released the NO after light irradiation to the same extent as a drug aqueous solution at the same pH. The drug dispersed in this gel also penetrated into the stratum corneum skin layer, and the nitro-ruthenium complex present in the skin was able to release the NO after light stimuli, suggesting the potential use of this formulation as a topical NO delivery system. (C) 2010 Elsevier B.V. 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.

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.

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.

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.

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.

Assessing flow in physical activity: The Flow State Scale-2 and Dispositional Flow Scale-2

The Flow State Scale-2 (FSS-2) and Dispositional Flow Scale-2 (DFS-2) are presented as two self-report instruments designed to assess flow experiences in physical activity. Item modifications were made to the original versions of these scales in order to improve the measurement of some of the flow dimensions. Confirmatory factor analyses of an item identification and a cross-validation sample demonstrated a good fit of the new scales. There was support for both a 9-first-order factor model and a higher order model with a global flow factor. The item identification sample yielded mean item loadings on the first-order factor of .78 for the FSS-2 and .77 for the DFS-2. Reliability estimates ranged from .80 to .90 for the FSS-2, and .81 to .90 for the DFS-2. In the cross-validation sample, mean item loadings on the first-order factor were .80 for the FSS-2, and .73 for the DFS-2. Reliability estimates ranged between .80 to .92 for the FSS-2 and .78 to .86 for the DFS-2. The scales are presented as ways of assessing flow experienced within a particular event (FSS-2) or the frequency of flow experiences in chosen physical activity in general (DFS-2).

Rescuing Wolbachia have been overlooked ...

Comment: Wolbachia, intracellular bacteria transmitted through the egg, have been estimated to infect more than 16% of all insect species, as well as other arthropods. They distort their hosts' reproduction, inducing parthenogenesis, feminization and cytoplasmic incompatibility. This favours the reproduction of infected female hosts at the expense of uninfected females. Here we show that several Wolbachia strains that cannot generate modifications in host sperm can still rescue the modifications caused by other strains as long as the two strains are sufficiently closely related.

A review of the use of the basic cation saturation ratio and the "ideal" soil

The use of 'balanced' Ca, Mg, and K ratios, as prescribed by the basic cation saturation ratio (BCSR) concept, is still used by some private soil-testing laboratories for the interpretation of soil analytical data. This review aims to examine the suitability of the BCSR concept as a method for the interpretation of soil analytical data. According to the BCSR concept, maximum plant growth will be achieved only when the soil’s exchangeable Ca, Mg, and K concentrations are approximately 65 % Ca, 10 % Mg, and 5 % K (termed the ‘ideal soil’). This ‘ideal soil’ was originally proposed by Firman Bear and co-workers in New Jersey (USA) during the 1940s as a method of reducing luxury K uptake by alfalfa (Medicago sativa L.). At about the same time, William Albrecht, working in Missouri (USA), concluded through his own investigations that plants require a soil with a high Ca saturation for optimal growth. Whilst it now appears that several of Albrecht’s experiments were fundamentally flawed, the BCSR (‘balanced soil’) concept has been widely promoted, suggesting that the prescribed cationic ratios provide optimum chemical, physical, and biological soil properties. Our examination of data from numerous studies (particularly those of Albrecht and Bear, themselves) would suggest that, within the ranges commonly found in soils, the chemical, physical, and biological fertility of a soil is generally not influenced by the ratios of Ca, Mg, and K. The data do not support the claims of the BCSR, and continued promotion of the BCSR will result in the inefficient use of resources in agriculture and horticulture.