Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid

Lys49-Phospholipase A(2) (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region pert-nits quaternary structural transitions between open and closed membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure [1]. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78Angstrom, (ii) MjTX-II/STE complex at a resolution of 1.8 Angstrom and (W) BthTX-I/DMPC complex at 2.72Angstrom. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (W) and using using a Synchrotron Radiation Source (Laboratorio Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).

Influence of Blood Contamination on Bond Strength of a Self-etching Adhesive to Dental Tissues

Purpose: The aim of this study was to detect the influence of (1) storage period of heparinized blood, (2) type of blood and presence of contaminant, (3) application mode of cleansing agents, and (4) efficacy of cleansing agents on contaminated enamel and dentin during the adhesion process of a one-step adhesive system. Materials and Methods: One hundred four human molars were sectioned into halves along the long axis for enamel and dentin tests. Heparinized and fresh blood were obtained from the same donor, applied and dried to maintain a layer of dry blood on the top of samples. The cleansing agents used were hydrogen peroxide, anionic detergent, and antiseptic solution. A one-step adhesive system (Clearfil S3 Bond) was applied on the dental surface, and composite resin cylinders were built up using Tygon tubing molds. After 24 h, the mu SBS test (1 mm/min) and fracture analysis were performed. Results: There was no statistically significant difference in bond strength values regarding the storage period of heparinized blood and the types of blood. Groups without contamination presented higher bond strengths than contaminated groups. The application mode of the cleansing agents had no influence on bond strength results. There was no statistically significant difference among cleansing agents and they were as effective as a water stream in counteracting the effect of blood contamination. Conclusion: Heparinized blood can be used as a contaminant for up to one week, and it is a reliable procedure to standardize the contaminant. The cleansing agents can be used without friction. A water stream is sufficient to remove blood contamination from dental tissues, before the application of a one-step adhesive system.

Influence of oil contamination on in vitro bond strength of bonding agents to dental substrates

Purpose: To evaluate the influence of cleaning procedures (pumice, anionic detergent and both procedures together) on the tensile bond strength of etch-and-rinse and self-etch adhesive systems to bovine enamel and dentin in vitro. Methods: Eighty non-carious, bovine incisors were extracted, embedded in acrylic resin to obtain enamel/dentin specimens. Flat bonding surfaces were obtained by grinding. Groups were divided according to substrate (enamel or dentin), adhesive system [etch-and-rinse, Adper Single Bond 2 (SB) or self-etch, Clearfil Protect Bond (PB)]; and cleaning substances (pumice, anionic detergent and their combination). The teeth were randomly divided into 20 groups (n=8): G1 - Enamel (E) + SB; G2 -E + oil (O) + SB; G3 - E + O + Pumice (P) + SB; G4 - E + O + Tergentol (T) + SB; G5 - E + O + P + T + SB; G6 - E + PB; G7 - E + O + PB; G8 - E + O + P + PB; G9 - E + O + T + PB; GIO - E + O + P + T + PB; G11 - Dentin (D) + SB; G12 D + SB + O; G13 - D + SB + O + P; G14 - D + SB + O + T; G15 - D + SB + O + P + T; G16 - D + PB; G17 - D + O + PB +; G18 - D + O + P + PB; G19 - D + O + T + PB; G20 - D + O + P + T + PB. Specimens were contaminated with handpiece oil for 5 seconds before bonding. Adhesive systems and resin composite were applied according to manufacturers` instructions. Specimens were tested in tension after 24 hours of immersion using a universal testing machine at a crosshead speed of 0.5 mm/minute. Bond strengths were analyzed with ANOVA. Failure sites were observed and recorded. Results: Tensile bond strength in MPa were: G1 (23.6 +/- 0.9); G2 (17.3 +/- 2.2); G3 (20.9 +/- 0.9); G4 (20.6 +/- 0.5); G5 (18.7 +/- 2.3); G6 (23.0 +/- 1.0); G7 (21.5 +/- 2.4); G8 (19.9 +/- 1.3); G9 (22.1 +/- 1.2); G10 (19.1 +/- 1.2); G11 (18.8 +/- 1.3); G12 (15.7 +/- 2.1); G13 (17.8 +/- 3.3); G14 (15.3 +/- 2.9); G15 (15.6 +/- 1.9); G16 (14.7 +/- 2.3); G17 (5.5 +/- 0.9); G18 (19.3 +/- 1.8); G19 (15.6 +/- 1.6); G20 (20.3 +/- 3.9). Statistical analysis showed that the main factors substrate and cleaning were statistically significant, as well as the triple interaction between factors of variance. However, the factor adhesive system did not show statistical difference. Oil contamination reduced bond strengths, being less detrimental to enamel than to dentin. Etch-and-rinse (SB) and two-step self-etch (PB) systems had similar bond strengths in the presence of oil contamination. For etch-and-rinse (SB), the cleaning procedures were able to clean enamel, but dentin was better cleaned by pumice. When self-etch (PB) system was used on enamel, anionic detergent was the best cleaning substance, while on dentin the tested procedures were similarly efficient.

Stability-indicating comparative methods using mekc and lc for determination of olmesartan medoxomil

A stability-indicating method using MEKC was validated for the analysis of olmesartan medoxomil in tablets. Successful separation was achieved using a fused silica capillary (40 cm x 50 µm i.d.); background electrolyte consisted of a combination of 10 mmol L-1 borate buffer and 5 mmol L-1 anionic detergent sodium dodecyl sulfate (95:5; v/v) pH 6.5; hydrodynamic mode at 50 mBar for 5 s; 25 kV separation voltage at 25 ºC; and column temperature 25 ºC with detection at 257 nm. The proposed method, validated following ICH guidelines, was applied to the determination of this antihypertensive with good results compared with an LC method.

Opposite roles of apolipoprotein E in normal brains and in Alzheimer’s disease

We have characterized the interaction between apolipoprotein E (apoE) and amyloid β peptide (Aβ) in the soluble fraction of the cerebral cortex of Alzheimer’s disease (AD) and control subjects. Western blot analysis with specific antibodies identified in both groups a complex composed of the full-length apoE and Aβ peptides ending at residues 40 and 42. The apoE–Aβ soluble aggregate is less stable in AD brains than in controls, when treated with the anionic detergent SDS. The complex is present in significantly higher quantity in control than in AD brains, whereas in the insoluble fraction an inverse correlation has previously been reported. Moreover, in the AD subjects the Aβ bound to apoE is more sensitive to protease digestion than is the unbound Aβ. Taken together, our results indicate that in normal brains apoE efficiently binds and sequesters Aβ, preventing its aggregation. In AD, the impaired apoE–Aβ binding leads to the critical accumulation of Aβ, facilitating plaque formation.

Biochemical Effects of Anionic, Cationic and Non Ionic Surfactants on a Tropical Teleost Oreochromis Mossambicus(Peters) and a Marine Cyanobacterium Synechocystis Salina Wislouch

The present work is a base line attempt to investigate and assess the toxicities of three surfactants viz. anionic sodium dodecyl sulfate (SDS), non ionic Triton X-1OO (TX-IOO) and cationic cetyl trimethyl ammonium bromide (CTAB). These compounds represent simple members of the often neglected group of aquatic pollutants i.e. the anionic alkyl sulfates, non ionics and the cationics. These compounds are widely used In plastic industry, pesticide/herbicide formulations, detergents, oil spill dispersants, molluscicides etc. The test organisms selected for the present study are the cyanobacterium Synechocystis salina Wislouch representing a primary producer in the marine environment and a fresh water adapted euryhaline teleost Oreochromis mossambicus (peters) at the consumer level of the ecological pyramid. The fish species, though not indigenous to our country, is now found ubiquitously in fresh water systems and estuaries. Also it is highly resistant to pollutants and has been suggested as an indicator of pollution in tropical region .

Counterion effects on the structure and solubilization properties of anionic detergents. Role of hydrophobic cations in stabilizing micelles and attenuating their solvent properties

A series of alkyl sulfate detergents has been investigated in the presence of the cations Na +, methylviologen(2+) (MV 2+), 4-(cyanomethyl)pyridinium(1+) (CMP +), and tetramethylammonium (TMA +). The binding of these ions to the aqueous micellar assemblies has been measured through studies of luminescence quenching with the extramicellar probe, RuL 34-, where L = 4,4′-dicarboxy-2,2′-bipyridine. A general comparison of the alkyl sulfate aggregates with the nonquenching cations Na + and TMA + shows that the latter ion reduces the critical micelle concentration but at the same time depresses the ability of the detergent assemblies to bind or solubilize the hydrophobic quencher cations MV 2+ or CMP +. The reduced binding ability of the TMA + aggregates compared to that of the corresponding Na + soaps shows up largely in the form of a reduced favorable ΔS° for the solubilization in the case of the former. The results are in accord with a picture of the TMA + micelle as being more stable and more disordered than the corresponding assembly with Na + as the counterion. © 1989 American Chemical Society.

Determination Of Detergent And Dispensant Additives In Gasoline By Ring-oven And Near Infrared Hypespectral Imaging.

A method using the ring-oven technique for pre-concentration in filter paper discs and near infrared hyperspectral imaging is proposed to identify four detergent and dispersant additives, and to determine their concentration in gasoline. Different approaches were used to select the best image data processing in order to gather the relevant spectral information. This was attained by selecting the pixels of the region of interest (ROI), using a pre-calculated threshold value of the PCA scores arranged as histograms, to select the spectra set; summing up the selected spectra to achieve representativeness; and compensating for the superimposed filter paper spectral information, also supported by scores histograms for each individual sample. The best classification model was achieved using linear discriminant analysis and genetic algorithm (LDA/GA), whose correct classification rate in the external validation set was 92%. Previous classification of the type of additive present in the gasoline is necessary to define the PLS model required for its quantitative determination. Considering that two of the additives studied present high spectral similarity, a PLS regression model was constructed to predict their content in gasoline, while two additional models were used for the remaining additives. The results for the external validation of these regression models showed a mean percentage error of prediction varying from 5 to 15%.

Clinical evaluation and induced corneal vascularization study by native and anionic collagen membranes in rabbits corneas

PURPOSE: To evaluate the corneal vascularization (CV) and the clinical aspects induced by interlamellar graft with native (NCM) and anionic (ACM) collagen membranes in rabbits corneas. METHODS: An interlamellar graft with a 0.25 x 0.25 cm NCM (group 1) or ACM (group 2) fragment was performed in the right eye (treated eye). In the left eye, an estromal tunnel was done (control eye). Sixteen rabbits were used, and they were subdivided into two experimental groups of eight animals each. The clinical evaluation was performed at the 1st, 3rd, 7th, 15th and 30th postoperative days. Corneal vascularization analysis was performed after 30 days by the Images Analizator System Leica Qwin-550®. RESULTS: After 7 days, corneal vascularization was observed at about 2.25 ± 0.71 mm (NCM) and at about 1.0 ± 1.69 mm (ACM), respectively, from the limbus in direction to the central cornea. After 15 days, CV increased in both groups (5.25 ± 1.03 mm - NCM; 2.0 ± 2.39 mm - ACM) and then progressively decreased until day 30 (2.25 ± 2.10 mm - NCM; 0.75 ± 2.12 mm - ACM). The statistical analysis indicated that the averages of the distances from the limb vessels to the grafts observed after 7 and 15 days had not differed statistically (p=0.17), and after 15 and 30 postoperative days had a tendency to differ statistically (p=0.09). The control eyes did not present any changes. CONCLUSION: The interlamellar graft with native and anionic collagen membranes induced corneal vascularization when applied to rabbit corneas, but anionic collagen membrane induced a smaller corneal vascularization when compared to native collagen membrane. Although further studies are required, the results found in this study demonstrated the usefulness of interlamellar graft with native and anionic collagen membranes in keratoplasties. These membranes consists in one more graft option for the surgical treatment of corneal repair in rabbits and others animals, when other forms of medical and surgical treatment are not effective.

Treatment of linear alkylbenzene sulfonate in a horizontal anaerobic immobilized biomass reactor

Linear alkylbenzene sulfonate (LAS) is an anionic surfactant widely used to manufacture detergents and found in domestic and industrial wastewater. LAS removal was evaluated in a horizontal anaerobic immobilized biomass reactor. The system was filled with polyurethane foam and inoculated with sludge that was withdrawn from an up flow anaerobic sludge blanket reactor that is used to treat swine wastewater. The reactor was fed with easily degradable substrates and a solution of commercial LAS for 313 days. The hydraulic retention time applied was 12 h. The system was initially operated without detergent and resulted to 94% reduction of demand. The mass balance in the system indicated that the LAS removal efficiency was 45% after 180 days. From the 109th day to the 254th day, a removal efficiency of 32% was observed. The removal of LAS was approximately 40% when 1500 mg of LAS were applied in the absence of co-substrates suggesting that the LAS molecules were used selectively. Microscopic analyses of the biofilm revealed diverse microbial morphologies and denaturing gradient gel electrophoresis profiling showed variations in the total bacteria and sulfate-reducing bacteria populations. 16S rRNA sequencing and phylogenetic analyses demonstrated that members of the order Clostridiales were the major components of the bacterial community in the last step of the reactor operation. (c) 2009 Elsevier Ltd. All rights reserved.

Degradation of detergent (linear alkylbenzene sulfonate) in an anaerobic stirred sequencing-batch reactor containing granular biomass

This study aimed to determine the efficiency of an anaerobic stirred sequencing-batch reactor containing granular biomass for the degradation of linear alkylbenzene sulfonate (LAS), a surfactant present in household detergent. The bioreactor was monitored for LAS concentrations in the influent, effluent and sludge, pH, chemical oxygen demand, bicarbonate alkalinity, total solids, and volatile solids. The degradation of LAS was found to be higher in the absence of co-substrates (53%) than in their presence (24-37%). Using the polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE), we identified populations of microorganisms from the Bacteria and Archaea domains. Among the bacteria, we identified uncultivated populations of Arcanobacterium spp. (94%) and Opitutus spp. (96%). Among the Archaea, we identified Methanospirillum spp. (90%), Methanosaeta spp. (98%), and Methanobacterium spp. (96%). The presence of methanogenic microorganisms shows that LAS did not inhibit anaerobic digestion. Sampling at the last stage of reactor operation recovered 61 clones belonging to the domain bacteria. These represented a variety of phyla: 34% shared significant homology with Bacteroidetes, 18% with Proteobacteria, 11% with Verrucomicrobia, 8% with Fibrobacteres, 2% with Acidobacteria, 3% with Chlorobi and Firmicutes, and 1% with Acidobacteres and Chloroflexi. A small fraction of the clones (13%) were not related to any phylum. Published by Elsevier Ltd.

Insulin-regulatable phosphoproteins in 3T3-L1 adipocytes form detergent-insoluble complexes not associated with caveolin

Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin-regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, som: of these intermediates, including IRS-1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high-speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3-L1 adipocytes. A large proportion of adipocyte phosphoproteins co-fractionated in the HSP fraction. In an attempt to resolve insulin-regulatable phosphoproteins, we subjected P-32-labeled subcellular fractions to two-dimensional gel electrophoresis (2-DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X-100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin-regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent-insoluble signaling complexes.

Analysis of Detergent-Insoluble and Whole Cell Lysate Fractions of Resting Neutrophils Using High-Resolution Mass Spectrometry

Neutrophilic granulocytes play a major role in the initiation and resolution of the inflammatory response, and demonstrate significant transcriptional and translational activity. Although much was known about neutrophils prior to the introduction of proteomics, the use of MS-based methodologies has provided an unprecedented tool to confirm and extend previous findings. In the present study, we performed a Gel-LC-MS/MS analysis of neutrophil detergent insoluble and whole cell lysate fractions of resting neutrophils. We achieved a set of identifications through the use of high-resolution mass spectrometry and validation of its data. We identified a total of 1249 proteins with a wide range of intensities from both detergent-insoluble and whole cell lysate fractions, allowing a mapping of proteins such as those involved in intracellular transport (Rab and Sec family proteins) and cell signaling (S100 proteins). These results represent the most comprehensive proteomic characterization of resting human neutrophils to date, and provide important information relevant for further studies of the immune system in health and disease. The methods applied here can be employed to help us understand how neutrophils respond to various physiologic and pathophysiologic conditions and could be extended to protein quantitation after cell activation.

Substitution of crude cell wall for neutral detergent fibre in the equations of the Cornell Net Carbohydrate and Protein System that predict carbohydrate fractions: application to sunflower (Helianthus annulus L.)

Prediction of carbohydrate fractions using equations from the Cornell Net Carbohydrate and Protein System (CNCPS) is a valuable tool to assess the nutritional value of forages. In this paper these carbohydrate fractions were predicted using data from three sunflower (Helianthus annuus L.) cultivars, fresh or as silage. The CNCPS equations for fractions B(2) and C include measurement of ash and protein-free neutral detergent fibre (NDF) as one of their components. However, NDF lacks pectin and other non-starch polysaccharides that are found in the cell wall (CW) matrix, so this work compared the use of a crude CW preparation instead of NDF in the CNCPS equations. There were no differences in the estimates of fractions B, and C when CW replaced NDF; however there were differences in fractions A and B2. Some of the CNCPS equations could be simplified when using CW instead of NDF Notably, lignin could be expressed as a proportion of DM, rather than on the basis of ash and protein-free NDF, when predicting CNCPS fraction C. The CNCPS fraction B(1) (starch + pectin) values were lower than pectin determined through wet chemistty. This finding, along with the results obtained by the substitution of CW for NDF in the CNCPS equations, suggests that pectin was not part of fraction B(1) but present in fraction A. We suggest that pectin and other non-starch polysaccharides that are dissolved by the neutral detergent solution be allocated to a specific fraction (B2) and that another fraction (B(3)) be adopted for the digestible cell wall carbohydrates.