Search results for " permeability"
showing 10 items of 341 documents
Streptolysin O: the C-terminal, tryptophan-rich domain carries functional sites for both membrane binding and self-interaction but not for stable oli…
2001
AbstractStreptolysin O belongs to the class of thiol-activated toxins, which are single chain, four-domain proteins that bind to membranes containing cholesterol and then assemble to form large oligomeric pores. Membrane binding involves a conserved tryptophan-rich sequence motif located within the C-terminally located domain 4. In contrast, sites involved in oligomerization and pore formation have been assigned to domains 1 and 3, respectively. We here examined the functional properties of domain 4, which was recombinantly expressed with an N-terminal histidine tag for purification and an additional cysteine residue for covalent labeling. The fluorescently labeled fragment readily bound to…
Toxicity and mode of action of Bacillus thuringiensis Cry proteins in the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre)
2006
ABSTRACT Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with 125 I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, an…
Assessment of Escherichia coli B with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cell function.
2002
Background Flow cytometry has become a choice methodology for microbiological research. However, functional cytometric assays in live bacteria are still limited. This is due, in part, to the cell wall impairing penetration of vital dyes in bacteria, thus imposing permeabilization procedures. These manipulations may affect cell physiology, provoke cell aggregation or lysis, and they are time-consuming. Escherichia coli B strains have been used for mutagenic assays because of an altered lipopolysaccharide that provokes increased membrane permeability. We assessed the use of these strains as possible alternatives for flow cytometric assays to avoid the permeabilization steps. Methods Suspensio…
Lipid Bilayer Interactions of Peptidic Supramolecular Polymers and Their Impact on Membrane Permeability and Stability.
2020
The synthesis and physicochemical characterization of supramolecular polymers with tunable assembly profiles offer exciting opportunities, involving the development of new biomedical carriers. Because synthetic nanocarriers aim to transport substances across or toward cellular membranes, we evaluated the interactions of amphiphilic peptide-based supramolecular polymers with lipid bilayers. Here, we focused on nanorod-like supramolecular polymers, obtained from two C3-symmetric dendritic peptide amphiphiles with alternating Phe/His sequences, equipped with a peripheral tetraethylene glycol dendron (C3-PH) or charged ethylenediamine end groups (C3-PH+). Triggered by pH changes, these amphiphi…
Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature
2007
ABSTRACT The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the c…
Superoxide generation by human neutrophils induced by low doses of Escherichia coli hemolysin.
1991
Escherichia coli hemolysin (Hly) was isolated from bacterial culture supernatants by polyethylene glycol precipitation and centrifugation in glycerol density gradients. The toxin preparations contained less than 1 mol of lipopolysaccharide per 10 mol of protein, and they had no fatty acids. The capacity of purified hemolysin to stimulate superoxide anion production in polymorphonuclear leukocytes was monitored kinetically in a lumimeter by using the lucigenin assay and was correlated with the kinetics of transmembrane pore formation. When applied to leukocytes suspended in protein-free buffer, very low concentrations (0.02 to 0.1 HU/ml) of the toxin strongly stimulated the production of sup…
Optimization of the Ussing chamber setup with excised rat intestinal segments for dissolution/permeation experiments of poorly soluble drugs.
2016
AbstractContext: Prediction of the in vivo absorption of poorly soluble drugs may require simultaneous dissolution/permeation experiments. In vivo predictive media have been modified for permeation experiments with Caco-2 cells, but not for excised rat intestinal segments.Objective: The present study aimed at improving the setup of dissolution/permeation experiments with excised rat intestinal segments by assessing suitable donor and receiver media.Methods: The regional compatibility of rat intestine in Ussing chambers with modified Fasted and Fed State Simulated Intestinal Fluids (Fa/FeSSIFmod) as donor media was evaluated via several parameters that reflect the viability of the excised in…
Characterization of the Epithelial Permeation Enhancing Effect of Basic Butylated Methacrylate Copolymer—In Vitro Studies
2008
Membrane destabilizing properties and increased efflux of doxorubicin from liposomes caused by basic butylated methacrylate copolymer (BBMC), better known under its commercial trade name EUDRAGIT E, have been described in the scientific literature. Here, we investigated the effect of BBMC on suspended and filter-grown Caco2 cells with respect to apical-to-basal transport and membrane permeabilization using transport assays, trypan blue exclusion assay, measurements of transepithelial electrical resistance (TEER), confocal laser scanning microscopy, and transmission electron microscopy. The effect of inhibiting protein phosphatase 2A (PP2A) by okadaic acid was investigated by measuring TEER,…
Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gra…
1998
Streptolysin O (SLO) is a bacterial exotoxin that binds to cell membranes containing cholesterol and then oligomerizes to form large pores. Along with rings, arc-shaped oligomers form on membranes. It has been suggested that each arc represents an incompletely assembled oligomer and constitutes a functional pore, faced on the opposite side by a free edge of the lipid membrane. We sought functional evidence in support of this idea by using an oligomerization-deficient, non-lytic mutant of SLO. This protein, which was created by chemical modification of a single mutant cysteine (T250C) with N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid, formed hybrid oligomers with active SLO on memb…
Indicaxanthin inhibits NADPH oxidase (NOX)-1 activation and NF-κB-dependent release of inflammatory mediators and prevents the increase of epithelial…
2014
Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1β, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1β brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signal…