Direct Visualization Of The Action Of Triton X-100 On Giant Vesicles Of Erythrocyte Membrane Lipids.

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.

Fast And Direct Na And K Determination In Table, Marine, And Low-sodium Salts By X-ray Fluorescence And Chemometrics.

X-ray fluorescence (XRF) is a fast, low-cost, nondestructive, and truly multielement analytical technique. The objectives of this study are to quantify the amount of Na(+) and K(+) in samples of table salt (refined, marine, and light) and to compare three different methodologies of quantification using XRF. A fundamental parameter method revealed difficulties in quantifying accurately lighter elements (Z < 22). A univariate methodology based on peak area calibration is an attractive alternative, even though additional steps of data manipulation might consume some time. Quantifications were performed with good correlations for both Na (r = 0.974) and K (r = 0.992). A partial least-squares (PLS) regression method with five latent variables was very fast. Na(+) quantifications provided calibration errors lower than 16% and a correlation of 0.995. Of great concern was the observation of high Na(+) levels in low-sodium salts. The presented application may be performed in a fast and multielement fashion, in accordance with Green Chemistry specifications.

Análise das metodologias diretas e indiretas para a contagem de células somáticas no leite de cabras hígidas

A particularidade da secreção láctea caprina, do tipo apócrina, diferente da secreção merócrina da vaca, leva a erros de interpretação durante a realização de técnicas de avaliação da celularidade do leite de fêmeas desta espécie. Portanto, o presente trabalho teve o objetivo de determinar a contagem de células somáticas pelo método indireto California Mastitis Test (CMT), e por métodos diretos, incluindo a contagem por citometria de fluxo e a contagem microscópica direta, através da coloração de verde de metil e pironina-Y, além de comparar os métodos de contagem celular. Foram analisadas 102 amostras de 51 fêmeas caprinas, das raças Saanen, Parda Alpina e Toggenburg, criadas no Estado de São Paulo. Os animais foram categorizados segundo a fase da lactação, exame físico da glândula mamária e exame do leite. As amostras foram colhidas, após a realização do exame Califórnia Mastitis Test, em duas alíquotas, uma destinada à contagem celular automática e a outra, a contagem microscópica direta, utilizando-se o corante verde de metil e pironina- Y. De acordo com os diferentes escores do CMT, observou- se 74,5% de amostras negativas, 8,8% de amostras com escore traços, 8,8% de amostras ligeiramente positivas (+), 6,8% de amostras fracamente positivas (++) e 0,9% de amostras fortemente positivas (+++). Os valores medianos das contagens de células somáticas presentes no leite de cabras, avaliadas através de contador automático e microscopia direta, e analisadas de acordo com os diferentes escores do CMT, foram, respectivamente, 181.000, 578.000, 628.000, 1.421.500 e 5.542.000 células/mL de leite e 74.991, 271.396, 71.420, 640.995 e 5.049.394 células/ mL de leite, nos escores negativo, traços, +, ++ e +++. Os valores medianos obtidos através da contagem de células somáticas pelo método automático e microscópico direto, de acordo com as fases de lactação foram de 159.500, 508.000 e 277.500 células/mL de leite, e 62.493, 89.275 e 146.411. A correlação obtida entre a contagem celular automática e microscópica direta foi de 88%. A partir dos resultados observados pode-se concluir que existe diferença na contagem celular determinada através do método automático e microscópico sendo este último o mais adequado para a determinação da celularidade no leite de cabras.

Direct drinking water treatment by spiral-wound ultrafiltration membranes

This paper presents the results of a research on direct drinking water treatment through an ultrafiltration pilot plant unit using spiral-wound membranes (3500 MWCO). The source of water is the Guarapiranga Reservoir, an eutrophicated water body located in the metropolitan region of Sao Paulo, Brazil. The data were collected during a period of almost 3400 h, from August 2005 to January 2006. The main objective of the study was to evaluate the membrane production capacity and contaminant removal efficiency. It was verified that the system was able to produce a high quality permeate with a flow close to the specified by the membrane manufacturer. The average permeate flow was 19.7 L.h(-1).m(-2), at 467 kPa and 25 degrees C, with a global water recovery of almost 85%. The removal efficiencies for TOC, UV light absorption, and turbidity were 85%, 56%, and 95%, respectively. The results provide substantial evidence of the technical feasibility of spiral-wound UF membranes for direct drinking water treatment from euthrophicated sources, as an alternative for conventional drinking water treatment systems.

CALCINED SLUDGE SINTERING EVALUATION BY HEATING MICROSCOPY THERMAL ANALYSIS

Aiming the use of the sewage sludge produced in one of the largest Brazilian wastewater treatment stations as a raw material for the ceramic industry, the sintering process of the ashes produced from its calcination was evaluated by heating microscopy thermal analysis (HMTA). From the microprocessed images, a method was developed to obtain HMTA dimensional change curves as a function of temperature, equivalent to those usually obtained from dilatometers or by thermomechanical analysis (TMA). The final product after sintering at 1050 degrees C, characterized by X-ray fluorescence spectrometry, scanning electron microscopy and X-ray dispersive energy, indicates the presence of a vitreous phase containing phosphorus, which explains the good sintering properties of the studied calcined sludge, as shown from its HMTA dimensional change curve.

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of Sao Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were reisolated in populations ranging from 10(6) to 10(9) CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms.

Quorum sensing detected by atomic force microscopy imaging of corrals surrounding multicellular arrangement of bacteria

Connectivity of the glycocalyx covering of small communities of Acidithiobacillus ferrooxidans bacteria deposited on hydrophilic mica plates was imaged by atomic force microscopy. When part of the coverage was removed by water rinsing, an insoluble structure formed by corrals surrounding each individual bacterium was observed. A collective ring structure with clustered bacteria (>= 3) was observed, which indicates that the bacteria perceived the neighborhood in order to grow a protective structure that results in smaller production of exopolysaccharides material. The most surprising aspect of these collective corral structures was that they occur at a low bacterial cell density. The deposited layers were also analyzed by confocal Raman microscopy and shown to contain polysaccharides, protein, and glucoronic acid.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

P>Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Parana, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff`s method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 mu l DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Direct Zincation of Functionalized Aromatics and Heterocycles by Using a Magnesium Base in the Presence of ZnCl(2)

A wide range of polyfunctional aryl and heteroaryl zinc reagents were efficiently prepared in THF by using (TMP)(2)Mg center dot 2LiCl (TMP = 2,2,6,6-tetramethylpiperamidyl) in the presence of ZnCl(2). The possible pathways of this metalation procedure as well as possible reactive intermediates are discussed. This experimental protocol expands the tolerance of functional groups and allows an efficient zincation of sensitive heterocycles such as quinoxaline or pyrazine. The zincated arenes and heteroarenes react with various electrophiles providing the expected products in 60-95 % yield.

Direct and Simultaneous Determination of Cd Cu, and Se in Blood Samples by Graphite Furnace Atomic Absorption Spectrometry

A graphite furnace atomic absorption spectrometric method is proposed for the direct and simultaneous determination of Cd, Cu, and Se in human blood. Samples were diluted 1:10 (v/v) in 0.5% (v/v) HNO(3) + 0.5% (v/v) Triton X-100 solution. For 12 mu L injected sample volume + 5 mu L, of 1000 mg L(-1) Pd(NO(3))(2) + 3 mu L of 1000 mg L(-1) Mg(NO(3))(2), the calculated characteristic masses (mo) were 0.9 pg Cd, 16 pg Cu, and 39 pg Se, which are close to those mo values for single-element conditions for THGA furnace (1.3 pg Cd, 17 pg Cu, and 45 pg Se). Calibration curves with linear correlations better than 0.999 were obtained. The limits of detection (LOD) were 0.03 mu g L(-1) Cd, 0.075 mu g L(-1) Cu and 0.3 mu g L(-1) Se, and the relative standard deviations (n= 12) were 2.5%, 0.3%, and 1.5%, respectively. The method was applied for Cd, Cu, and Se determination in 10 human blood samples and the results were in agreement at the 95% confidence level with those obtained by inductively coupled plasma mass spectrometry. Concentrations of analytes in the selected blood samples varied from 1.7 to 3.2 mu g L(-1) Cd, 700 to 921.7 mu g L(-1) Cu, and from 68.6 to 350 mu g L(-1) Se. The accuracy of the proposed method was also evaluated by an addition-recovery experiment and recoveries of Cd, Cu, and Se added to blood samples ranged from 99-109%, 91-103%,and 93-103%, respectively.

Effect of Annatto on Micronuclei Induction by Direct and Indirect Mutagens in HepG2 Cells

Annatto (AN), a natural food colorant rich in carotenoids, has been reported as being an effective antioxidant, but little is known about its potential chemopreventive properties. In this Study, we evaluated the ability of AN to protect human hepatoma cells (HepG2) from micronucleus (MN) induction against three different mutagens: benzo(a)pyrene (B(a)P), doxorubicin (DXR), and methyl methanesulfonate (MMS). In an attempt to clarify the possible mechanism of anti mutagenicity of AN, three protocols of treatment were applied (pretreatment; simultaneous treatment, and post-treatment with AN following treatment with the mutagens). Also, cells exposed only to AN were assayed for cytotoxicity and mutagenicity. A dosage up to 10 mu g/ml of AN was devoid of mutagenic activity. Protective effects were seen on micronuclei induced by B(a)P and DXR using pre and simultaneous treatment, but AN had no significant effect on MN induction by MMS in any of the protocols. Our results also show that exposure of cells to concentrations of AN higher than 10 mu g/ml decreased cell viability. Taken together, our findings indicate that AN presents antimutagenic activity in vitro, but its protective effect is dependent on the mutagen and on type of treatment suggesting its potential use as a chemopreventive agent. Environ. Mol. Mutagen. 50:808-814, 2009. (C) 2009 Wiley-Liss, Inc.

The 1.1 angstrom resolution crystal structure of [Tyr(15)]EpI, a novel alpha-conotoxin from Conus episcopatus, solved by direct methods

Conotoxins are valuable probes of receptors and ion channels because of their small size and highly selective activity. alpha-Conotoxin EpI, a 16-residue peptide from the mollusk-hunting Conus episcopatus, has the amino acid sequence GCCSDPRCNMNNPDY(SO3H)C-NH2 and appears to be an extremely potent and selective inhibitor of the alpha 3 beta 2 and alpha 3 beta 4 neuronal subtypes of the nicotinic acetylcholine receptor (nAChR). The desulfated form of EpI ([Tyr(15)]EpI) has a potency and selectivity for the nAChR receptor similar to those of EpI. Here we describe the crystal structure of [Tyr(15)]EpI solved at a resolution of 1.1 Angstrom using SnB. The asymmetric unit has a total of 284 non-hydrogen atoms, making this one of the largest structures solved de novo try direct methods. The [Tyr(15)]EpI structure brings to six the number of alpha-conotoxin structures that have been determined to date. Four of these, [Tyr(15)]EpI, PnIA, PnIB, and MII, have an alpha 4/7 cysteine framework and are selective for the neuronal subtype of the nAChR. The structure of [Tyr(15)]EpI has the same backbone fold as the other alpha 4/7-conotoxin structures, supporting the notion that this conotoxin cysteine framework and spacing give rise to a conserved fold. The surface charge distribution of [Tyr(15)]EpI is similar to that of PnIA and PnIB but is likely to be different from that of MII, suggesting that [Tyr(15)]EpI and MII may have different binding modes for the same receptor subtype.

Tubular system volume changes in twitch fibres from toad and rat skeletal muscle assessed by confocal microscopy

The volume of the extracellular compartment (tubular system) within intact muscle fibres from cane toad and rat was measured under various conditions using confocal microscopy. Under physiological conditions at rest, the fractional volume of the tubular system (t-sys(Vol)) was 1.38 +/- 0.09% (n = 17),1.41 +/- 0.09% (n = 12) and 0.83 +/- 0.07% (n = 12) of the total fibre volume in the twitch fibres from toad iliofibularis muscle, rat extensor digitorum longus muscle and rat soleus muscle, respectively. In toad muscle fibres, the t-sys(Vol) decreased by 30% when the tubular system was fully depolarized and decreased by 15% when membrane cholesterol was depleted from the tubular system with methyl-beta-cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 mum. There was also an increase by 30% and a decrease by 25% in t-sys(Vol) when toad fibres were equilibrated in solutions that were 2.5-fold hypertonic and 50% hypotonic, respectively. When the changes in total fibre volume were taken into consideration, the t-sys(Vol) expressed as a percentage of the isotonic fibre volume did actually decrease as tonicity increased, revealing that the tubular system in intact fibres cannot be compressed below 0.9% of the isotonic fibre volume. The results can be explained in terms of forces acting at the level of the tubular wall. These observations have important physiological implications showing that the tubular system is a dynamic membrane structure capable of changing its volume in response to the membrane potential, cholesterol depletion and osmotic stress but not when the sarcomere length is changed in resting muscle.

Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta: Liposomes fusion and/or lysis investigated by fluorescence and atomic force microscopy

Three dermaseptins, DS 01, DD K, and DD L, were compared with respect to their structural features and interactions with liposomes. Circular dichroic spectra at alcohols of different chain lengths revealed that DS 01 has the higher helicogenic potential in hydrophobic media. Binding of DS 01, DD K, and DD L to liposomes induced significant blue shifts of the emission spectra of the single tryptophan located at position 3 of all sequences indicating association of the peptides with bilayers. Kinetics evaluation of atomic force microscopy images evidenced the strong fusogenic activity of DS 01 whereas DD K and DD L showed increased lytic activities. (C) 2007 Elsevier Inc. All rights reserved.