Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Biomembranes (v.1714, #2)
Effect of “helper lipid� on lipoplex electrostatics by Danielle Hirsch-Lerner; Ming Zhang; Hagit Eliyahu; Marylin E. Ferrari; Carl J. Wheeler; Yechezkel Barenholz (pp. 71-84).
Lipoplexes, which are complexes between cationic liposomes (L+) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA—each with and without helper lipid (cholesterol or DOPE). Electrical surface potential ( Ψ0) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl- sn-glycero-3-phosphoethanolamine- N-carboxyfluorescein (CFPE) and 1,2-dioleyl- sn-glycero-3-phosphoethanolamine- N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Ψ0 determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Ψ0 indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Ψ0 determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA−/L+ charge ratios 0.5–1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.
Keywords: Abbreviations; Chol; cholesterol; CFPE; 1,2-dioleyl-; sn; -glycero-3-phosphoethanolamine-; N; -(carboxyfluorescein); C17HC; 4-heptadecyl-7-hydroxycoumarin; DC-Chol; 3β-[; N; -(; N; ′,; N; ′-dimethyl-aminoethane)-carbamoyl]-cholesterol; DMRIE; N; -(1-(2,3-dimyristyloxypropyl)-; N; ,; N; -dimethyl-(2-hydroxyethyl) ammonium bromide; DNA; −; /L; +; mole charge ratio of DNA negatively-charged phosphate to positively-charged lipid; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DOPE; 1,2-dioleoyl-; sn; -glycero-3-phosphoethanolamine; DOSPA; 2,3-dioleyloxy-; N; -[2(sperminecarboxamido)-ethyl]-; N; ,; N; -dimethyl-1-propanaminium trifluoro acetate; DOTAP; N; -(1-(2,3-dioleoyloxy)propyl)-; N,N,N; -trimethylammonium chloride; HC-DOPE; 1,2-dioleyl-; sn; -glycero-3-phosphoethanolamine-; N; -(7-hydroxycoumarin); HEPES; N; -(2-hydroxyethyl)-piperazine-; N; ′-(2-ethanesulfonic acid); L; +; positively charged lipid; LUV; large (≥ 100 nm) unilamellar vesicles; ODN; oligonucleotide; TMADPH; 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene; UHV; unsized heterogeneous vesiclesCationic lipid; DNA; Surface potential; Hydroxycoumarin; Carboxyfluorescein; Ionic strength
Structure–affinity relationship in the interactions of human organic anion transporter 1 with caffeine, theophylline, theobromine and their metabolites by Mitsuru Sugawara; Takahiro Mochizuki; Yoh Takekuma; Katsumi Miyazaki (pp. 85-92).
It is well known that human organic anion transporter 1 (hOAT1) transports many kinds of drugs, endogenous compounds, and toxins. However, little is known about the structure–affinity relationship. The aim of this study was to elucidate the structure–affinity relationship using a series of structurally related compounds that interact with hOAT1. Inhibitory effects of xanthine- and uric acid-related compounds on the transport of p-aminohippuric acid were examined using CHO-K1 cells stably expressing hOAT1. The order of potency for the inhibitory effects of xanthine-related compounds on PAH uptake was 1-methyl derivative>7-methyl derivative>3-methyl derivative≒xanthine>1,3,7-trimethyl derivative (caffeine). The order of potency of the inhibition was 1,3,7-trimethyluric acid>1,3-dimethyluric acid>1,7-dimethyluric acid>1-methyluric acid>uric acid. A significant correlation between inhibitory potency and lipophilicity of the tested uric acid-related compounds was observed. The main determinant of the affinity of xanthine-related compounds is the position of the methyl group. On the other hand, lipophilicity is the main determinant of the affinity of uric acid-related compounds.
Keywords: hOAT1; Caffeine; Theophylline; Structure–affinity relationship; Interaction
Study of the surface morphology of a cholesteryl tethering system for lipidic bilayers by L. Blasi; D. Pisignano; F. Di Benedetto; G. Maruccio; G. Ciccarella; A. Maffei; G. Vasapollo; R. Cingolani; R. Rinaldi (pp. 93-102).
The immobilization of functional molecules embedded in lipidic membranes onto inorganic substrates is of great interest for numerous applications in the fields of biosensors and biomaterials. We report on the preparation and the morphological characterization of a tethering system for lipidic bilayers, which is based on cholesteryl derivatives deposited on hydrophilic surfaces by self-assembling and microcontact printing techniques. The investigation of the structural properties of the realized films by atomic, lateral, and surface potential microscopy allowed us to assess the high quality of the realized cholesteryl layers.
Keywords: Cholesteryl derivative; Lipid bilayer; Scanning probe microscopy; Soft lithography
Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy by Lars Thomas; Holger A. Scheidt; Andrea Bettio; Daniel Huster; Annette G. Beck-Sickinger; Klaus Arnold; Olaf Zschörnig (pp. 103-113).
Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the α-helical part of NPY (amino acids 13–36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1–12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface Δ G supports the preferential interaction of NPY with negatively charged membranes.
Keywords: Abbreviations; 5-doxyl PC; 1-palmitoyl-2-stearoyl-(5-doxyl)-; sn; -glycero-3-phosphocholine; AMPSO; 3-[(1,1-Dimethyl-2-hydroxy-ethyl)amino]-2-hydroxy-propanesulfonic acid; CROX; potassium chromium oxalate (K; 3; Cr(C; 2; O; 4; ); 3H; 2; O); DPC; dodecylphosphocholine; LUV; large unilamellar vesicles; MAS; magic-angle spinning; MES; 2-[N-morpholino]ethanesulfonic acid; MLV; multilamellar vesicles; NOE; nuclear Overhauser effect; NPY; neuropeptide Y; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoserine; PS; phosphatidylserine; TOAC; 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acidLiposome; TOAC; 1; H MAS NMR; Peptide lipid interaction; CROX; Membrane partitioning; Amphipathic helix
Quantification and distribution of big conductance Ca2+-activated K+ channels in kidney epithelia by Morten Grunnet; Anders Hay-Schmidt; Dan A. Klaerke (pp. 114-124).
Big conductance Ca2+ activated K+ channels (BK channels) is an abundant channel present in almost all kind of tissue. The accurate quantity and especially the precise distribution of this channel in kidney epithelia are, however, still debated. The aim of the present study has therefore been to examine the presence of BK channels in kidney epithelia and determine the actual number and distribution of these channels. For this purpose, a selective peptidyl ligand for BK channels called iberiotoxin or the radiolabeled double mutant analog125I-IbTX-D19Y/Y36F has been employed. The presence of BK channels were determined by a isotope flux assay where up to 44% of the total K+ channel activity could be inhibited by iberiotoxin indicating that BK channels are widely present in kidney epithelia. Consistent with these functional studies,125I-IbTX-D19Y/Y36F binds to membrane vesicles from outer cortex, outer medulla and inner medulla with Bmax values (in fmol/mg protein) of 6.8, 2.6 and 21.4, respectively. These studies were performed applying rabbit kidney epithelia tissue. The distinct distribution of BK channels in both rabbit and rat kidney epithelia was confirmed by autoradiography and immunohistochemical studies. In cortical collecting ducts, BK channels were exclusively located in principal cells while no channels could be found in intercalated cells. The abundant and distinct distribution in kidney epithelia talks in favor for BK channels being important contributors in maintaining salt and water homeostasis.
Keywords: Transepithelial transport; Binding studies; Immunohistochemistry; BK channels
Resveratrol inhibits polyphosphoinositide metabolism in activated platelets by Beata Olas; Barbara Wachowicz; Holm Holmsen; Miriam H. Fukami (pp. 125-133).
The effects of resveratrol ( trans-3,4′,5-trihydroxystilbene) on activation responses and the polyphosphoinositide metabolism in human blood platelets have been studied. Resveratrol partially inhibited secretory responses (liberation of dense granule nucleotides and lysosomal acid hydrolases), microparticle formation and protein phosphorylations induced by thrombin. The effects of resveratrol on phosphoinositide metabolites, phosphatidate (PtdOH), phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4(5)- P), phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5- P 2), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4- P 2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5- P 3) were monitored in blood platelets prelabelled with [32P]Pi. Resveratrol not only inhibited the marked increase in levels of PtdOH in platelets activated by thrombin (0.1 U/ml) but it decreased the steady state levels of the other polyphosphoinositide metabolites. The distribution of32P in phosphoinositides in activated platelets was consistent with inhibition of CDP-DAG inositol transferase and a weak inhibition of PtdIns-4(5)- P kinase. These observations show that resveratrol has a profound effect on phospholipids, particularly on polyphosphoinositide metabolism, and may decrease the amount of PtdIns-4,5- P 2 available for signalling in these cells.
Keywords: Blood platelet; Polyphosphoinositide signalling; Resveratrol
Mechanism of inhibition of proton: Dipeptide co-transport during chronic enteritis in the mammalian small intestine by Uma Sundaram; Sheik Wisel; Steven Coon (pp. 134-140).
Amino acids, a critical energy source for the intestinal epithelial cells, are more efficiently assimilated in the normal intestine via peptide co-transporters such as proton:dipeptide co-transport (such as PepT1). Active uptake of a non-hydrolyzable dipeptide (glycosarcosine) was used as a substrate and PepT1 was found to be present in normal villus, but not crypt cells. The mRNA for this transporter was also found in villus, but not crypt cells from the normal rabbit intestine. PepT1 was significantly reduced in villus cells also diminished in villus cell brush border membrane vesicles both from the chronically inflamed intestine. Kinetic studies demonstrated that the mechanism of inhibition of PepT1 during chronic enteritis was secondary to a decrease in the affinity of the co-transporter for the dipeptide without an alteration in the maximal rate of uptake ( Vmax). Northern blot studies also demonstrated unaltered steady state mRNA levels of this transporter in the chronically inflamed intestine. Proton dipeptide transport is found in normal intestinal villus cells and is inhibited during chronic intestinal inflammation. The mechanism of inhibition is secondary to altered affinity of the co-transporter for the dipeptide.
Keywords: Abbreviations; BBM; Brush Border Membrane; BBMV; Brush Border Membrane Vesicles; EDTA; ethylene diamine tetraacetic acidProton; Dipeptide; Chronic enteritis; Amino acid absorption; Regulation of amino acid transport
Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label: The effect of cholesterol by Paresh C. Dave; Nisreen A. Nusair; Johnson J. Inbaraj; Gary A. Lorigan (pp. 141-151).
X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, Smol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.
Keywords: Magnetically aligned bilayers; Order parameter; Spin-label; Bicelle; EPR