Preparation of Liposomes Containing Oleanolic Acid via Micelle-to-Vesicle Transition

2007 ◽  
Vol 7 (11) ◽  
pp. 3944-3948 ◽  
Author(s):  
Hyung Seok Kang ◽  
Ji-Eun Park ◽  
Young-Jin Lee ◽  
Ih-Seop Chang ◽  
Yong-Il Chung ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Bilayers ◽  
Oleanolic Acid ◽  
Potassium Salt ◽  
Mechanical Treatment ◽  
Micelle Formation ◽  
Liposomal Suspension ◽  
Aqueous Mixture ◽  
Soluble State ◽  
Homogenization Step

Micelle-to-vesicle transition method was used to make liposomes containing oleanolic acid. First, the solubilization of potassium salt of oleanolic acid at basic condition by micelle formation was confirmed. Using the soluble state of oleanolic acid at basic condition, liposomes containing oleanolic acid was prepared by adjusting pH. After making homogeneous aqueous mixture of potassium salt of oleanolic acid and lecithin in basic condition, the solution was neutralized to produce the lecithinbased liposomes that contain oleanolic acid inside the lipid bilayers. The optimal loading of oleanolic acid to lecithin (about 25 mole%) was found to exist to produce liposomal suspension of small size without homogenization step. Electron microscopy and dynamic light scattering studies showed that the narrowly distributed, reconstituted oleanolic acid-containing liposomes were prepared without severe mechanical treatment.

scholarly journals Fracture faces of frozen membranes: 50th anniversary

2016 ◽  
Vol 27 (3) ◽  
pp. 421-423
Author(s):  
Daniel Branton
Keyword(s):  
Electron Microscopy ◽  
Lipid Bilayers ◽  
Fracture Process ◽  
Relative Proportion ◽  
50Th Anniversary ◽  
Biological Membranes ◽  
Bilayer Membrane ◽  
Biological Cells ◽  
Membrane Surfaces ◽  
Freeze Etching

In 1961, the development of an improved freeze-etching (FE) procedure to prepare rapidly frozen biological cells or tissues for electron microscopy raised two important questions. How does a frozen cell membrane fracture? What do the extensive face views of the cell’s membranes exposed by the fracture process of FE tell us about the overall structure of biological membranes? I discovered that all frozen membranes tend to split along weakly bonded lipid bilayers. Consequently, the fracture process exposes internal membrane faces rather than either of the membrane’s two external surfaces. During etching, when ice is allowed to sublime after fracturing, limited regions of the actual membrane surfaces are revealed. Examination of the fractured faces and etched surfaces provided strong evidence that biological membranes are organized as lipid bilayers with some proteins on the surface and other proteins extending through the bilayer. Membrane splitting made it possible for electron microscopy to show the relative proportion of a membrane’s area that exists in either of these two organizational modes.

Bioavailability of Paediatric Mixtures Containing Phenoxymethylpenicillin Calcium or Potassium Salt

1982 ◽  
Vol 10 (5) ◽  
pp. 379-382 ◽  
Author(s):  
Kari Soininen ◽  
Hannu Allonen ◽  
Juhani Posti
Keyword(s):  
Potassium Salt ◽  
Calcium Salt ◽  
Healthy Adult ◽  
Peak Plasma ◽  
Peak Plasma Concentration ◽  
Salt Mixture ◽  
Time To Peak ◽  
Aqueous Mixture ◽  
The Mean ◽  
Adult Volunteers

The bioavailability of the calcium and potassium salts of Phenoxymethylpenicillin (dose 38,000 I.U./kg) was investigated in eight healthy adult volunteers. Administration of the calcium salt as an aqueous oral mixture resulted in a mean peak plasma concentration of 8·52 mg/l (SD 1·96) and that of the potassium salt mixture in a concentration of 8·40 mg/ml (SD 2·61), p > 0·1. The median time-to-peak levels were 0·75 h and 1·0 h, respectively (p > 0·1). The mean AUC for the calcium salt mixture was 16·94 mg·h/l (SD 3·31) and for the potassium salt 15·84 mg·h/l (SD 4·76), p > 0·09. These findings confirm that an aqueous mixture of calcium phenoxymethylpenicillin is equivalent to a mixture of potassium Phenoxymethylpenicillin.

Spectroscopic and biophysical approaches for studying the structure and function of the P-glycoprotein multidrug transporter

1998 ◽  
Vol 76 (5) ◽  
pp. 695-708 ◽  
Author(s):  
Frances J Sharom ◽  
Ronghua Liu ◽  
Yolanda Romsicki
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Multidrug Resistance ◽  
Fluorescence Spectroscopy ◽  
Lipid Bilayers ◽  
Drug Transport ◽  
Circular Dichroism Spectroscopy ◽  
P Glycoprotein ◽  
Infra Red ◽  
Circular Dichroïsm

Multidrug resistance is a serious obstacle to the successful chemotherapeutic treatment of many human cancers. A major cause of multidrug resistance is the overexpression of a 170-kDa plasma membrane protein, known as P-glycoprotein, which appears to function as an ATP-driven efflux pump with a very broad specificity for hydrophobic drugs, peptides, and natural products. P-Glycoprotein is a member of the ABC superfamily and is proposed to consist of two homologous halves, each comprising six membrane-spanning segments and a cytosolic nucleotide binding domain. In recent years, P-glycoprotein has been purified and functionally reconstituted into lipid bilayers, where it retains both ATPase and drug transport activity. The availability of purified active protein has led to substantial advances in our understanding of the molecular structure and mechanism of action of this unique transporter. This review will focus on the recent application of fluorescence spectroscopy, infra-red spectroscopy, circular dichroism spectroscopy, electron microscopy, and other biophysical techniques to the study of P-glycoprotein structure and function.Key words: multidrug resistance, P-glycoprotein, fluorescence spectroscopy, infra-red spectroscopy, circular dichroism spectroscopy, differential scanning calorimetry, electron microscopy.

scholarly journals Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release

2021 ◽  
Author(s):  
Kevin C Courtney ◽  
Yueqi Li ◽  
Jason D Vevea ◽  
Zhenyong Wu ◽  
Zhao Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Bilayers ◽  
Force Microscopy ◽  
Anionic Phospholipids ◽  
Synaptic Vesicle Exocytosis ◽  
Lysine Residues ◽  
Vesicle Exocytosis ◽  
Synaptotagmin 1 ◽  
Self Association ◽  
Aqueous Conditions

Synaptotagmin-1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer, however biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures (~180 nm) were reduced to smaller rings (~30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in: a) clamping spontaneous release, and b) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.

Screening for Fibrin Specific Monoclonal Antibodies: The Development of a New Procedure

1992 ◽  
Vol 68 (01) ◽  
pp. 048-053 ◽  
Author(s):  
P M Tymkewycz ◽  
L J Creighton-Kempsford ◽  
D Hockley ◽  
P J Gaffney
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Chemical Composition ◽  
Cross Reactivity ◽  
The Other ◽  
Screening Procedure ◽  
Screening Procedures ◽  
Soluble State ◽  
Scanning Electron

SummaryThe acquisition of monoclonal antibodies specific for human fibrin has been impaired by the similarity in chemical composition between fibrinogen and fibrin and the conformational difference between immobilised and soluble fibrinogen. Five monoclonal antibodies (mabs) with a known affinity for fibrin have been subjected to screening procedures which involved the presentation of different forms of both fibrinogen and fibrin to the test mabs. It was observed by scanning electron microscopy that dried fibrin (denoted fibrin D), immobilised on the wells of PVC plates was morphologically similar to the fibrin found in human clots whereas PVC-immobilised fibrin monolayers (fibrin M) and a homogenised form of fibrin (fibrin FF) presented two very different morphological appearances. It was shown that lack of cross reactivity of a mab with an antigen (e.g. fibrinogen) was validly demonstrated only when both mab and antigen were present in the soluble state. These findings have allowed the generation of a screening procedure which involves the use of fibrin D on PVC plates in conjunction with whole human plasma incubated with the test antibody. This screening procedure has been validated using two mabs, one of which has an exclusive fibrin affinity while the other has a broad spectrum crossreactivity with both fibrinogen and fibrin. This procedure would ensure the acquisition of all the five fibrin-specific mabs used in this study while other less reliable screening procedures might not.

The occurrence of lipidic particles in lipid bilayers as seen by 31P NMR and freeze-fracture electron-microscopy

1979 ◽  
Vol 555 (2) ◽  
pp. 200-209 ◽  
Author(s):  
B. De Kruijff ◽  
A.J. Verkley ◽  
C.J.A. Van Echteld ◽  
W.J. Gerritsen ◽  
C. Mombers ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Bilayers ◽  
31P Nmr ◽  
Freeze Fracture ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

scholarly journals A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers

1998 ◽  
Vol 329 (3) ◽  
pp. 571-577 ◽  
Author(s):  
E. Yuri KORCHEV ◽  
C. Lindsay BASHFORD ◽  
Cecilia PEDERZOLLI ◽  
A. Charles PASTERNAK ◽  
J. Peter MORGAN ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Membrane ◽  
Molecular Mass ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Bivalent Cations ◽  
Ring Structures ◽  
Ion Conducting ◽  
Small Channels

Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433 → Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433 → Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433 → Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433 → Phe. Electron microscopy revealed that Trp-433 → Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433 → Phe variant. We conclude that the change Trp-433 → Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.

Perturbation of lecithin bilayer structure by globoside

1976 ◽  
Vol 54 (3) ◽  
pp. 209-218 ◽  
Author(s):  
D. O. Tinker ◽  
L. Pinteric ◽  
J. C. Hsia ◽  
R. P. Rand
Keyword(s):  
Electron Microscopy ◽  
Phase Transition ◽  
Lipid Bilayers ◽  
Esr Spectroscopy ◽  
Repulsive Force ◽  
Spectroscopic Techniques ◽  
Bilayer Structure ◽  
X Ray Diffraction ◽  
X Ray ◽  
Spin Resonance

The ultrastructure of aggregates formed by mixtures of pig erythrocyte lecithin, cholesterol and globoside in aqueous systems was studied by electron microscopy and X-ray diffraction. Globoside and lecithin in up to equimolar amounts formed a lamellar mesophase, although the structure of the lamellae was perturbed. Mixtures containing excess globoside formed complex tubular or reticular aggregates. Cholesterol appeared to promote mixing of lecithin and globoside. The flexibility gradient of the hydrocarbon (hc) region of the lipid bilayers was studied using electron spin resonance (esr) spectroscopy of various nitroxide-labelled stearic acid probes. Globoside in equimolar amounts greatly perturbed the order parameters of lecithin bilayers, reducing the fluidity of the hc region and flattening the flexibility gradient near the polar (p) surface. The effect of globoside on lecithin–cholesterol bilayers was not so pronounced, since the latter was already more ordered than lecithin bilayers. A phase transition of pure globoside at 55 °C, involving 'melting' of the hc chains was also detected using X-ray and esr spectroscopic techniques. The interbilayer spacing, dw, of equimolar lecithin–globoside lamellar phase increased by 42% from that of lecithin bilayers, indicating that the glycolipid p group may increase the net repulsive force between bilayers, as was previously predicted theoretically.

scholarly journals Structure and Electrophysiological Properties of the YscC Secretin from the Type III Secretion System of Yersinia enterocolitica

2004 ◽  
Vol 186 (14) ◽  
pp. 4645-4654 ◽  
Author(s):  
Peter Burghout ◽  
Ria van Boxtel ◽  
Patrick Van Gelder ◽  
Philippe Ringler ◽  
Shirley A. Müller ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Outer Membrane ◽  
Lipid Bilayers ◽  
Yersinia Enterocolitica ◽  
Sodium Dodecyl ◽  
Angular Power Spectrum ◽  
Electrophysiological Properties ◽  
Type Iii ◽  
Scanning Transmission

ABSTRACT YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.

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