Search results for "erythrocytes"
showing 10 items of 218 documents
Expression of Active Streptolysin O in Escherichia coli as a Maltose-Binding-Protein-Streptolysin-O Fusion Protein. The N-Terminal 70 Amino Acids are…
1996
Streptolysin 0 (SLO) is the prototype of a family of cytolysins that consists of proteins which bind to cholesterol and form very large transmembrane pores. Structure/function studies on the pore-forming cytolysin SLO have been complicated by the proteolytic inactivation of a substantial portion of recombinant SLO (rSLO) expressed in Escherichia coli. To overcome this problem, translational fusions between the E. coli maltose-binding protein (MBP) gene and SLO were constructed, using the vectors pMAL-p2 and pMAL-c2. MBP-SLO fusion proteins were degraded if secreted into the E. coli periplasm, but intact, soluble MBP-SLO fusion proteins were produced at high levels in the cytoplasm. Active S…
Transmembrane beta-barrel of staphylococcal alpha-toxin forms in sensitive but not in resistant cells.
1997
Staphylococcal α-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118–140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity coul…
Anti-Eryptotic Activity of Food-Derived Phytochemicals and Natural Compounds
2022
Human red blood cells (RBCs), senescent or damaged due to particular stress, can be removed by programmed suicidal death, a process called eryptosis. There are various molecular mechanisms underlying eryptosis. The most frequent is the increase in the cytoplasmic concentration of Ca2+ ions, later exposure of erythrocytes to oxidative stress, hyperosmotic shock, ceramide formation, stimulation of caspases, and energy depletion. Phosphatidylserine (PS) exposed by eryptotic RBCs due to interaction with endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor, causes the RBCs to adhere to vascular wall with consequent damage to the microcirculation. Eryptosis can be triggered by various xenobiotics…
Red Blood Cells Polarize Green Laser Light Revealing Hemoglobin's Enhanced Non-Fundamental Raman Modes
2014
In general, the first overtone modes produce weak bands that appear at approximately twice the wavenumber value of the fundamental transitions in vibrational spectra. Here, we report the existence of a series of enhanced non-fundamental bands in resonance Raman (RR) spectra recorded for hemoglobin (Hb) inside the highly concentrated heme environment of the red blood cell (RBC) by exciting with a 514.5 nm laser line. Such bands are most intense when detecting parallel-polarized light. The enhancement is explained through excitonic theory invoking a type C scattering mechanism and bands have been assigned to overtone and combination bands based on symmetry arguments and polarization measureme…
Dietary indicaxanthin from cactus pear (Opuntia ficus-indica L. Mill) fruit prevents eryptosis induced by oxysterols in a hypercholesterolaemia-relev…
2015
Toxic oxysterols in a hypercholesterolaemia-relevant proportion cause suicidal death of human erythrocytes or eryptosis. This process proceeds through early production of reactive oxygen species (ROS), release of prostaglandin (PGE2) and opening of PGE2-dependent Ca channels, membrane phosphatidylserine (PS) externalisation, and cell shrinkage. The present study was the first to reveal that a bioavailable phytochemical, indicaxanthin (Ind) from cactus pear fruit, in a concentration range (1·0–5·0 μM) consistent with its plasma level after a fruit meal, prevents PS externalisation and cell shrinkage in a dose-dependent manner when incubated with isolated healthy human erythrocytes exposed to…
Quantitative contributions of IgG, IgM and C3 to erythrophagocytosis and rosette formation by peritoneal macrophages, and anti-opsonin activity of de…
1976
In vitro phagocytosis by guinea pig peritoneal macrophages of immune complexes (EA) was shown to be dependent on IgG antibody in a dose-dependent fashion. C3b enhanced phagocytosis of EA at limited IgG antibody concentrations only. When IgM antibody was used for sensitization of sheep red blood cells (SRBC), phagocytosis and rosette formation did not occur in the absence of bound C3. The polyanion, dextran sulfate 500 (DS), was shown to depress both rosette formation and phagocytosis of EAIgG, C1423 and EAIgMC1423, as well as immune adherence of human group 0 erythrocytes and hemolytic activity of C3. This effect of DS was seen only when it was actually present in the incubation medium.
Evidence that clustered phosphocholine head groups serve as sites for binding and assembly of an oligomeric protein pore.
2006
High susceptibility of rabbit erythrocytes toward the pore-forming action of staphylococcal alpha-toxin correlates with the presence of saturable, high affinity binding sites. All efforts to identify a protein or glycolipid receptor have failed, and the fact that liposomes composed solely of phosphatidylcholine are efficiently permeabilized adds to the enigma. A novel concept is advanced here to explain the puzzle. We propose that low affinity binding moieties can assume the role of high affinity binding sites due to their spatial arrangement in the membrane. Evidence is presented that phosphocholine head groups of sphingomyelin, clustered in sphingomyelin-cholesterol microdomains, serve th…
Oxysterol mixture in hypercholesterolemia-relevant proportion causes oxidative stress-dependent eryptosis.
2014
Background/Aims: Oxysterol activity on the erythrocyte (RBC) programmed cell death (eryptosis) had not been studied yet. Effects of an oxysterol mixture in hyper-cholesterolemic-relevant proportion, and of individual compounds, were investigated on RBCs from healthy humans. Methods: Membrane phosphatidylserine (PS) externalization, calcium entry, ROS production, amino-phospholipid translocase (APLT) activity were evaluated by cytofluorimetric assays, cell volume from forward scatter. Prostaglandin PGE2 was measured by ELISA; GSH-adducts and lipoperoxides by spectrophotometry. Involvement of protein kinase C and caspase was investigated by inhibitors staurosporin, calphostin C, and Z-DEVD-FM…
Swellable microparticles containing Suprofen: evaluation of in vitro release and photochemical behaviour
1998
Suprofen, an anti-inflammatory drug was incorporated in polymer networks based on biocompatible macromolecules, such as alpha,beta-polyasparthydrazide (PAHy) and alpha,beta-poly(N-hydroxyethyl)-DL-aspartamide (PHEA) crosslinked by glutaraldehyde or gamma-rays, respectively. Swelling tests carried out in aqueous media showed that pH value affects the swelling degree of the prepared hydrogels. In vitro release tests were performed in simulated gastrointestinal fluids (pH 1/6.8) using the pH variation method and in phosphate-buffered saline, pH 7.4. Experimental data indicated that Suprofen was released in a sustained way both from PAHy and PHEA microparticles. Further, incorporation of Suprof…
Cysteine-Specific Radioiodination of Proteins with Fluorescein Maleimide
1997
A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label is then removed by gel-permeation chromatography. The procedure avoids exposition of the protein to oxidative conditions and does not require purification of the labeled carrier reagent. Suitability of the method for a given protein can be evaluated spectrophotometrically without employing radioactivity. It can be applied under denaturing condit…