Control of lipid membrane stability by cholesterol content.
(1/539)
Cholesterol has a concentration-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane potential difference modulation induced in vesicles when an external electric field is applied. The magnitude of this modulation is under the control of the membrane electrical permeability. When brought to a critical value by the external field, the membrane potential difference induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content. (+info)
Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.
(2/539)
Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies. (+info)
Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia.
(3/539)
Paroxysmal nocturnal hemoglobinuria (PNH), frequently occurring during suppressed hematopoiesis including aplastic anemia (AA), is a clonal disorder associated with an increased incidence of thrombotic events. Complement-mediated hemolysis, impairment of the fibrinolytic system, or platelet activation are thought to be responsible for the associated thrombotic risk. We investigated here the elevation of membrane-derived procoagulant microparticles in the blood flow of such patients. Elevated levels of circulating microparticles were in fact detected in both de novo PNH patients and AA subjects with a PNH clone, but not in those with AA without a PNH clone. The cellular origin of the microparticles was determined in PNH samples; most stemmed from platelets. Glycophorin A+ particles were rarely detected. Therefore, platelet activation, resulting in the dissemination of procoagulant phospholipids in the blood flow, could be one of the main causes for the elevated thrombotic risk associated with PNH. These observations suggest that shed membrane particles can be considered a valuable biological parameter for the assessment of possible thrombotic complications in patients with PNH. (+info)
The expression of human blood group antigens during erythropoiesis in a cell culture system.
(4/539)
Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3. (+info)
XRCC1 polymorphisms: effects on aflatoxin B1-DNA adducts and glycophorin A variant frequency.
(5/539)
Hereditary genetic defects in DNA repair lead to increased risk of cancer. Polymorphisms in several DNA repair genes have been identified; however, the impact on repair phenotype has not been elucidated. We explored the relationship between polymorphisms in the DNA repair enzyme, XRCC1 (codons 194, 280, and 399), and genotoxic end points measured in two populations: (a) placental aflatoxin B1 DNA (AFB1-DNA) adducts in a group of Taiwanese maternity subjects (n = 120); and (b) somatic glycophorin A (GPA) variants in erythrocytes from a group of North Carolina smokers and nonsmokers (n = 59). AFB1-DNA adducts were measured by ELISA, and erythrocyte GPA variant frequency (NN and NO) was assessed in MN heterozygotes with a flow cytometric assay. XRCC1 genotypes were identified by PCR-RFLPs. The XRCC1 399Gln allele was significantly associated with higher levels of both AFB1-DNA adducts and GPA NN mutations. Individuals with the 399Gln allele were at risk for detectable adducts (odds ratio, 2.4; 95% confidence interval, 1.1-5.4; P = 0.03). GPA NN variant frequency was significantly higher in 399Gln homozygotes (19.6 x 10(-6)) than in Gln/Arg heterozygotes (11.4 x 10(-6); P < 0.05) or Arg/Arg homozygotes (10.1 x 10(-6); P = 0.01). No significant effects were observed for other XRCC1 polymorphisms. These results suggest that the Arg399Gln amino acid change may alter the phenotype of the XRCC1 protein, resulting in deficient DNA repair. (+info)
A method for determining transmembrane helix association and orientation in detergent micelles using small angle x-ray scattering.
(6/539)
Solution small angle x-ray scattering can be used to study the association of transmembrane proteins solubilized in detergent micelles. We have used the alpha-helical transmembrane domain of the human erythrocyte glycophorin A (GpA) fused to the carboxyl terminus of monomeric staphylococcal nuclease (SN/GpA) as a model system for study. By matching the average electron density of the detergent micelles to that of the buffer solution, the micelle contribution to the small angle scattering vanishes, and the molecular weight and the radius of gyration of the proteins can be determined. SN/GpA has been found to dimerize in a zwitterionic detergent micelle, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), whose average electron density naturally matches the electron density of an aqueous buffer. The dimerization occurs through the transmembrane domains of GpA. With the aid of the nuclease domain scattering, the orientation of the helices within a dimer can be determined to be parallel by radius of gyration analysis. The association constant of a mutant (G83I) that weakens the GpA dimerization has been determined to be 24 microM in the DDMAB environment. The experimental methods established here could be used to apply solution small angle x-ray scattering to studying the association and interactions of other membrane proteins. (+info)
Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action.
(7/539)
To study the role of transmembrane (TM) domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent-resistant dimers through TM-TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the beta subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints. (+info)
Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance.
(8/539)
Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosphatidylinositol (GPI) anchors on host cell-surface structures. The nature of the receptors and the location of the receptor-binding sites on the toxin molecule were investigated using surface plasmon resonance. Aerolysin bound to the GPI-anchored proteins Thy-1, variant surface glycoprotein, and contactin with similar rate constants and affinities. Enzymatic removal of N-linked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin. Aerolysin is a bilobal protein, and both lobes were shown to be required for optimal binding. The large lobe by itself bound Thy-1 with an affinity that was at least 10-fold weaker than that of the whole toxin, whereas the small lobe bound the GPI-anchored protein at least 1000-fold more weakly than the intact toxin. Mutation analyses provided further evidence that both lobes were involved in GPI anchor binding, with certain single amino acid substitutions in either domain leading to reductions in affinity of as much as 100-fold. A variant with single amino acid substitutions in both lobes of the protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weakly to immobilized proaerolysin, suggesting that interactions with cell-surface carbohydrate structures other than GPI anchors may partially mediate toxin binding to host cells. (+info)