498 research outputs found
Coupling Optical and Electrical Measurements in Artificial Membranes: Lateral Diffusion of Lipids and Channel Forming Peptides in Planar Bilayers
Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K(+) channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules
Selective Adsorption and Chiral Amplification of Amino Acids in Vermiculite Clay -Implications for the origin of biochirality
Smectite clays are hydrated layer silicates that, like micas, occur naturally
in abundance. Importantly, they have readily modifiable interlayer spaces that
provide excellent sites for nanochemistry. Vermiculite is one such smectite
clay and in the presence of small chain-length alkyl-NH3Cl ions, forms
sensitive, 1-D ordered model clay systems with expandable nano-pore inter-layer
regions. These inter-layers readily adsorb organic molecules. N-propyl NH3Cl
vermiculite clay gels were used to determine the adsorption of alanine, lysine
and histidine by chiral HPLC. The results show that during reaction with fresh
vermiculite interlayers, significant chiral enrichment of either L- and
D-enantiomers occurs depending on the amino acid. Chiral enrichment of the
supernatant solutions is up to about 1% per pass. In contrast, addition to clay
interlayers already reacted with amino acid solutions resulted in little or no
change in D/L ratio during the time of the experiment. Adsorption of small
amounts of amphiphilic organic molecules in clay inter-layers is known to
produce Layer-by-Layer or Langmuir-Blodgett films. Moreover atomistic
simulations show that self-organization of organic species in clay interlayers
is important. These non-centrosymmetric, chirally active nanofilms may cause
clays to act subsequently as chiral amplifiers, concentrating organic material
from dilute solution and having different adsorption energetics for D- and
L-enantiomers. The additional role of clays in RNA oligimerization already
postulated by Ferris and others, together with the need for the organization of
amphiphilic molecules and lipids noted by Szostak and others, suggests that
such chiral separation by clays in lagoonal environments at normal biological
temperatures might also have played a significant role in the origin of
biochirality.Comment: 17 Pages, 2 Figures, 4 Table
Effects of Perfluorocarbons on surfactant exocytosis and membrane properties in isolated alveolar type II cells
<p>Abstract</p> <p>Background</p> <p>Perfluorocarbons (PFC) are used to improve gas exchange in diseased lungs. PFC have been shown to affect various cell types. Thus, effects on alveolar type II (ATII) cells and surfactant metabolism can be expected, data, however, are controversial.</p> <p>Objective</p> <p>The study was performed to test two hypotheses: (I) the effects of PFC on surfactant exocytosis depend on their respective vapor pressures; (II) different pathways of surfactant exocytosis are affected differently by PFC.</p> <p>Methods</p> <p>Isolated ATII cells were exposed to two PFC with different vapor pressures and spontaneous surfactant exocytosis was measured. Furthermore, surfactant exocytosis was stimulated by either ATP, PMA or Ionomycin. The effects of PFC on cell morphology, cellular viability, endocytosis, membrane permeability and fluidity were determined.</p> <p>Results</p> <p>The spontaneous exocytosis was reduced by PFC, however, the ATP and PMA stimulated exocytosis was slightly increased by PFC with high vapor pressure. In contrast, Ionomycin-induced exocytosis was decreased by PFC with low vapor pressure. Cellular uptake of FM 1-43 - a marker of membrane integrity - was increased. However, membrane fluidity, endocytosis and viability were not affected by PFC incubation.</p> <p>Conclusions</p> <p>We conclude that PFC effects can be explained by modest, unspecific interactions with the plasma membrane rather than by specific interactions with intracellular targets.</p
Cholesterol Corrects Altered Conformation of MHC-II Protein in Leishmania donovani Infected Macrophages: Implication in Therapy
Previously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of
class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activatio
Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol
Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol
Fabrication Principles and Their Contribution to the Superior In Vivo Therapeutic Efficacy of Nano-Liposomes Remote Loaded with Glucocorticoids
We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t1/2 ∼1 h), or a slow, zero-order release rate (t1/2 ∼ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies
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