Freeze-fracture Electron Microscopy Studies Research Articles

Polarization-Modulated Bent-Core Liquid Crystal Thin Films without Layer Undulation.

Spatial confinement is known to affect molecular organizations of soft matter. We present an important manifestation of this statement for thin films of bent-core smectic liquid crystals. Prior freeze-fracture transmission electron microscopy (FFTEM) studies carried out on nitro-substituted bent-core mesogens (n-OPIMB-NO_{2}) revealed an undulated smectic layer structure with an undulation periodicity of ∼8 nm. We report cryogenic TEM measurements on ∼100 nm thick 8-OPIMB-NO_{2} films. In contrast to FFTEM results, our studies show only density modulation with periodicity b=16.2 nm, and no smectic layer undulation. We show that the discrepancy between the FFTEM and cryogenic transmission electron microscopy (cryo-TEM) results can be attributed to the different sample thicknesses used in the experiments. FFTEM monitors cracked surfaces of a relatively thick (5-10 μm) frozen sample, whereas cryo-TEM visualizes the volume of a thin (0.1 μm) film that was quenched from its partially fluid phase. These results have importance in possible photovoltaics and organic electronics applications where submicron thin films are used.

Reformulation of etoposide with solubility-enhancing rubusoside

Etoposide (ETO), a widely used anti-cancer drug, is constrained by its low aqueous solubility and by side effects from both the drug and its solubilizing excipients. In this study, a recently discovered natural solubilizer rubusoside (RUB) was used to achieve the solubilization of ETO. Dynamic light scattering and freeze-fracture transmission electron microscopy studies showed that ETO and RUB formed ETO–RUB nanoparticles (∼6nm in diameter). The powder of ETO–RUB nanoparticles was completely reconstitutable in water and remained stable in this solution at 25 and 37°C for at least 24h. Under other physiologic conditions, ETO solution was clear and free of precipitation at 25°C, but underwent various structural transformations. In PBS and simulated intestinal fluid, RUB-solubilized ETO underwent epimerization and equilibrated to cis-ETO. In simulated gastric fluid, RUB-solubilized ETO degraded to 4′-demethylepipodophyllotoxin-beta-d-glucoside and 4′-demethylepipodophyllotoxin. Higher temperatures favored epimerization or degradation. Furthermore, a side-by-side comparison with DMSO-solubilized ETO confirmed that the RUB-solubilized ETO showed no significant differences in cytotoxicity in colon, breast and prostate cancer cell lines. RUB effectively solubilized and stabilized etoposide, which sets the stage for further toxicology, bioavailability, and efficacy investigations.

Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras

The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 "long" (M1) and "short" (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 degrees C indicated that OAPs diffuse slowly (D approximately 10(-12) cm(2)/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.

Structure of biodiesel based bicontinuous microemulsions for environmentally compatible decontamination: A small angle neutron scattering and freeze fracture electron microscopy study

Most toxic industrial chemicals and chemical warfare agents are hydrophobic and can only be solubilized in organic solvents. However, most reagents employed for the degradation of these toxic compounds can only be dissolved in water. Hence, microemulsions are auspicious media for the decontamination of a variety of chemical warfare agents and pesticides. They allow for the solubilization of both the lipophilic toxics and the hydrophilic reagent. Alkyl oligoglucosides and plant derived solvents like rapeseed methyl ester enable the formulation of environmentally compatible bicontinuous microemulsions. In the present article the phase behavior of such a microemulsion is studied and the bicontinuous phase is identified. Small angle neutron scattering (SANS) and freeze fracture electron microscopy (FFEM) measurements are used to characterize the structure of the bicontinuous phase and allow for an estimation of the total internal interface. Moreover, also the influence of the co-surfactant (1-pentanol) on the structural parameters of the bicontinuous phase is studied with SANS.

The effect of electrolyte on the encapsulation efficiency of vesicles formed by the nonionic surfactant, 2C 18E 12

Encapsulation efficiencies of vesicles formed by the nonionic surfactant 1,2-dioctadecyl- rac-glycerol-3- ω-methoxydodecylethylene glycol (abbreviated as 2C 18E 12) and its phospholipid counterpart, distearoylphosphatidylcholine (DSPC) at 298 K, were determined by the entrapment of the water-soluble dye, carboxyfluorescein (CF) to be 0.045 ± 0.001 and 0.03 ± 0.04 L mol −1 for 2C 18E 12 vesicles prepared using low osmolarity (270 m Osm) Krebs–Henseleit (K–H) buffer and a modified ‘high salt’ (1600 m Osm) variant of K–H buffer, respectively, and 0.64 ± 0.01 and 0.31 ± 0.04 L mol −1 for DSPC vesicles prepared under the same conditions and in the same buffers. Freeze fracture electron microscopy studies confirmed the presence of vesicles when 2C 18E 12 and DSPC were dispersed in water and both buffer solutions. Small angle neutron scattering (SANS) studies, using D 2O in place of H 2O, showed that when 2C 18E 12 vesicles were prepared in the ‘high salt’ variant of K–H buffer as opposed to K–H buffer or water, a higher proportion of multilamellar vesicles (MLV) were formed. Furthermore when prepared in the ‘high salt’ variant of K–H buffer, the 2C 18E 12 bilayers were thinner, and when present in the form of MLV exhibited a smaller layer of water separating the bilayers. However, even in the absence of electrolyte, 2C 18E 12 formed surprisingly thin bilayers due to the penetration of the polyoxyethylene chains into the hydrophobic chain region of the bilayer. Due to the dehydrating effect of the high concentration of electrolyte present in the ‘high salt’ variant of K–H, the polyoxyethylene head groups penetrated further into the hydrophobic region of the bilayer making the bilayer even thinner. In the case of the DSPC vesicles, although the SANS study showed an increase in the relative proportion of multilamellar to unilamellar vesicles when samples were prepared in the ‘high salt’ variant of K–H buffer, no differences were observed in the thickness and the d-spacing of the vesicle bilayers. Variable temperature turbidity measurements of 2C 18E 12, and DSPC vesicles prepared in water indicated phase changes at 320 ± 0.5 and 327 ± 0.5 K , respectively, and were unchanged when the ‘high salt’ variant of K–H buffer was used as hydrating medium. Taken together, these results suggest that a low phase transition temperature was not the reason for the poor entrapment efficiency of 2C 18E 12 vesicles but rather the very ‘thin’ hydrophobic barrier formed by the penetration of the polyoxyethylene chains into the hydrophobic region of the bilayer.

Investigation of surface-modified solid lipid nanocontainers formulated with a heterolipid-templated homolipid

There is increasing interest in the search for improved drug delivery systems with greater versatility. Consequently, many drug delivery systems have been studied. In this study, surface-modified lipid nanocontainers were formulated with a homolipid from Capra hircus (goat fat) templated with a heterolipid (Phospholipon 90G ®) which was also the surface modifier. The solid lipid nanocontainers (SLN) were formulated by hot high pressure homogenisation using increasing concentrations of polysorbate 80 as the mobile surfactant. Prior to SLN preparation, the templated homolipid was formulated by fusion to obtain a homogeneous lipid matrix, which was characterized using differential scanning calorimetry (DSC), polarized light microscopy (PLM) and wide angle X-ray diffraction (WAXD) to obtain its thermal and crystal characteristics. Isothermal heat conduction microcalorimetry (IMC) and freeze-fracture transmission electron microscopy (FFTEM) studies were carried out on the templated homolipid and SLN containing 1.0% (w/w) of polysorbate 80 to study their in situ crystallization kinetics and morphology, respectively. The formulated SLN were also subjected to time-resolved DSC, WAXD and particle size analyses for one month. The thermal and crystal characteristics were compared with those of the bulk lipid matrix (templated homolipid). Result of the particle size analysis indicated that the particles size remained roughly within the lower nanometer range after one month. FFTEM micrograph of the lipid matrices revealed lamellar sheets for Phospholipon 90G ® and layered triglyceride structures for the homolipid and Phospholipon 90G ®-templated homolipid. FFTEM micrograph of SLN revealed anisometric structures. PLM of the templated homolipid did not show, but goat fat (homolipid) alone showed slight growth in crystals with time. WAXD and DSC studies revealed minor increase in crystallinity of the new lipid matrix after one month and DSC also detected templation of homolipid by the heterolipid noted by the disappearance of the lower melting peak of the homolipid. However, for the SLN, WAXD results showed low crystalline particles while DSC only showed a very little endothermic process after one month of storage at 20°C. The implication of this finding is that progression of the SLN to highly ordered particles over time would not occur. This will be favourable for any incorporated drug as drug expulsion, due to increase in crystallinity, will not occur. Result obtained from analysis of the isothermal crystallization exotherms indicated that the templated homolipid and SLN1 containing 1.0% polysorbate 80 possess similar nucleation mechanisms and growth dimensions different from the pure homolipid. The SLN containing 0.5 and 1.0% polysorbate 80 possessed good properties and could prove to be good delivery systems for drugs for parenteral or ocular administration. The result of this study also shows a method of improving natural lipids for use in particulate drug delivery systems.

Proteocubosomes: Nanoporous Vehicles with Tertiary Organized Fluid Interfaces

Proteocubosomes are nanostructured open-nanochannel hierarchical fluid vehicles characterized by a cubic lattice periodicity of the lipid/protein supramolecular assembly (protein-loaded cubosomes). They are obtained here at very high hydration levels by a three-dimensional (3D) self-assembly process, which exploits a protein-directed 3D patterning and fragmentation to create a new, tertiary-level structural order of fluid lipid/water interfaces. Our freeze-fracture electron microscopy study reveals that the proteocubosome structures are built up by patterned assemblies of nanocubosomes, which comprise 3D nanoporous fracture surfaces throughout. Complex cubosomic architectures, involving arrays of nanodroplets (larger than 20 nm) inside the proteocubosome particles, are established at high resolution. The soft-matter hierarchical nanocompartment formations display internal aqueous pores belonging to the D-type lipid cubic lattice nanochannel system that is proven by synchrotron X-ray diffraction. The reported nanostructured fluid may give rise to novel applications in nanofluidic biomimetic devices, porous protein drug delivery vehicles, nanoscale enzymatic bioreactors, and protein-encapsulating fluid nanomaterials.

Freeze-fracture transmission electron microscopy studies on the self-assemblies of amphiphilic solutions

Self-assemblies of amphiphiles in solutions were investigated by using freeze-fracture transmission electron microscopy (FF-TEM). Especially, vesicles were characterized by FF-TEM and the transition of self-assemblies was determined. The stacked lamellar Lα-phase was prepared without shear forces by a chemical reaction. The stacked lamellar Lα-phase can be transformed into multilamellar vesicles by the shearing forces that occur when the stacked lamellar Lα-phase sample is turned upside down a few times. The multilamellar vesicles can also be transformed into unilamellar vesicles by high shearing forces. These transitions were demonstrated by FF-TEM measurements. Zn2+-induced vesicle formation in the single-chain surfactant solutions was first achieved.

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N, N-dioleyl- N, N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 °C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [ 31P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H II) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.

Slow Reorganization of Small Phosphatidylcholine Vesicles upon Adsorption of Amphiphilic Polymers

Static or dynamic light scattering measurements were performed in parallel, on dilute mixtures of DPPC/DPPA vesicles (typical radius 60 nm) and hydrophobically modified polymers. This technique gave evidence of the slow kinetics involved in both the reorganization of an adsorbed polymer layer and the membrane breakage. Hours, or sometimes days, were required in order to follow the variation of both the hydrodynamic radius and the scattering intensity at intermediate stages. Images of the intermediate species were collected using freeze-fracture electron microscopy (FFEM). Comparison of different polymers (of varying molecular weight or structure) revealed the prime importance of hydrophobicity on the disruption of membranes. Although the presence of a few percent of pendant alkyl chains along the polymer backbone induced adsorption to membranes, only the association with the more hydrophobic ones (>25 mol% of pendant octyl groups) resulted in small mixed objects of micellar size (radius about 10 nm). The drop of the mean radius of intermediate structures formed upon the vesicle breakage was also sensitive to temperature. A tentative mechanism was proposed on the basis of kinetics and FFEM studies.

Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents.

The amino acid homopolymers, poly-L-lysine and poly-L-ornithine, have been modified by the covalent attachment of palmitoyl and methoxypoly(ethylene glycol) (mPEG) residues to produce a new class of amphiphilic polymers-PLP and POP, respectively. These amphiphilic amino acid based polymers have been found to assemble into polymeric vesicles in the presence of cholesterol. Representatives of this new class of polymeric vesicles have been evaluated in vitro as nonviral gene delivery systems with a view to finding delivery systems that combine effective gene expression with low toxicity in vivo. In addition, the drug-carrying capacity of these polymeric vesicles was evaluated with the model drug doxorubicin. Chemical characterization of the modified polymers was carried out using (1)H NMR spectroscopy and the trinitrobenzene sulfonic acid (TNBS) assay for amino groups. The amphiphilic polymers were found to have an unreacted amino acid, palmitoyl, mPEG ratio of 11:5:1, and polymeric vesicle formation was confirmed by freeze-fracture electron microscopy and drug encapsulation studies. The resulting polymeric vesicles, by virtue of the mPEG groups, bear a near neutral zeta-potential. In vitro biological testing revealed that POP and PLP vesicle-DNA complexes are about one to 2 orders of magnitude less cytotoxic than the parent polymer-DNA complexes although more haemolytic than the parent polymer-DNA complexes. The polymeric vesicles condense DNA at a polymer:DNA weight ratio of 5:1 or greater and the polymeric vesicle-DNA complexes improved gene transfer to human tumor cell lines in comparison to the parent homopolymers despite the absence of receptor specific ligands and lysosomotropic agents such as chloroquine.

Electroperturbation of human stratum corneum fine structure by high voltage pulses: a freeze-fracture electron microscopy and differential thermal analysis study.

Application of high voltage pulses (HVP) to the skin has been shown to promote the transdermal drug delivery by a mechanism involving skin electroporation. The aim of this study was to detect potential changes in lipid phase and ultrastructure induced in human stratum corneum by various HVP protocols, using differential thermal analysis and freeze-fracture electron microscopy. Due to the time involved between the moment the electric field is switched off and the analysis, only "secondary" phenomena rather than primary events could be observed. A decrease in enthalpies for the phase transitions observed at 70 degrees C and 85 degrees C was detected by differential thermal analysis after HVP treatment. No changes in transition temperature could be seen. The freeze-fracture electron microscopy study revealed a dramatic perturbation of the lamellar ordering of the intercellular lipid after application of HVP. Most of the planes displayed rough surfaces. The lipid lamellae exhibited rounded off steps or a vanished stepwise order. There was no evidence for perturbation of the corneocytes content. In conclusion, the freeze-fracture electron microscopy and differential thermal analysis studies suggest that HVP application induces a general perturbation of the stratum corneum lipid ultrastructure.

A method for uni-directional reconstitution of human erythrocyte glucose transporter.

The glucose transporter of human erythrocytes (Glut1) was reconstituted into soybean phospholipid liposomes by a method of direct incorporation using the nonionic detergent n-octyl beta-D-glucopyranoside. The reconstituted proteoliposomes were proved to be intact and low ionic permeability. Freeze-fracture electron microscopy study showed that the diameter of the proteoliposomes was about 150 +/- 50 nm and the protein was randomly distributed. The kinetic parameters of the reconstituted transporter were: Km =16.23 mmol/L, Vmax = 34.48 nmol/sec x mg protein. Furthermore, about 90% of the glucose transporter in the reconstituted proteoliposomes were orientated inside-out. Until now it is a more efficient method for uni-directional reconstitution of Glut1 with good reproducibility and higher transport activity.

Structural and fusogenic properties of cationic liposomes in the presence of plasmid DNA

The structural and fusogenic properties of large unilamellar vesicles (LUVs) composed of the cationic lipid N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyl-3-phosphatidylethanotamine (DOPE) have been examined in the presence of pCMV5 plasmid and correlated with transfection potency. It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between DOTMA/DOPE (1:1) LUVs and DOTMA/1,2-dioleoyl-3-phosphatidylcholine (DOTMA/DOPC) (1:1) LUVs such that at a cationic lipid-to-DNA charge ratio of 3.0, approximately 80% fusion is observed. The anions citrate and chloride can also trigger fusion, but at much higher concentrations. Freeze-fracture electron microscopy studies demonstrate the tendency of cationic vesicles to form clusters at low pCMV5 content, whereas macroscopic fused aggregates can be observed at higher plasmid levels. 31P NMR studies of the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow "isotropic" 31P NMR resonances, whereas the corresponding DOPC containing systems exhibit much broader "bilayer" 31P NMR spectra. In agreement with previous studies, the transfection potency of the DOPE-containing systems is dramatically higher than for the DOPC-containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. Interestingly, it was found that the complexes could be separated by centrifugation into a pellet fraction, which exhibits superior transfection potencies, and a supernatant fraction. Again, the pellet fraction in the DOPE-containing system exhibits a significantly narrower 31P NMR resonance than the corresponding DOPC-containing system. It is suggested that the 31P NMR characteristics of complexes exhibiting higher transfection potencies are consistent with the presence of nonbilayer lipid structures, which may play a direct role in the fusion or membrane destabilization events vital to transfection.

Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete.

Previous freeze-fracture electron microscopy (EM) studies have shown that the outer membrane (OM) of Treponema pallidum contains sparse transmembrane proteins. One strategy for molecular characterization of these rare OM proteins involves isolation of T. pallidum OMs. Here we describe a simple and extremely gentle method for OM isolation based upon isopycnic sucrose density gradient ultracentrifugation of treponemes following plasmolysis in 20% sucrose. Evidence that T. pallidum OMs were isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical markers for periplasmic, cytoplasmic membrane, and cytosolic compartments, and (iii) freeze-fracture EM demonstrating that the putative OMs contained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the OMs contained a relatively small number of treponemal proteins, including several which did not appear to correspond to previously characterized T. pallidum antigens. Interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblotting and enhanced chemiluminescence. Compared with whole cells, T. pallidum OMs were deficient in cardiolipin, the major lipoidal antigen reactive with antibodies in syphilitic sera. Also noteworthy was that other lipoidal constituents of OMs, including the recently discovered glycolipids, did not react with human syphilitic sera. These latter observations suggest that the poor antigenicity of virulent T. pallidum is a function of both the lipid composition and the low protein content of its OM.

Water pathways across a reconstituted epithelial barrier formed by caco-2 cells: Effects of medium hypertonicity

Caco-2 cells, originated in a human colonic cancer, are currently used as model systems to study transepithelial transports. To further characterize their water permeability properties, clone P1 Caco-2 cells were cultured on permeable supports. At confluence, the transepithelial net water movement (Jw), mannitol permeability (Ps), and electrical resistance (R) were simultaneously measured. The observed results were correlated with transmission and freeze-fracture electron microscopy studies and compared with those obtained, in similar experimental conditions, in a typical mammalian epithelial barrier: the rabbit rectum. When the serosal solution was made hypertonic (50 mM polyethylene glycol-PEG), the spontaneously observed secretory Jw rapidly reversed, became absorptive and then stabilized. Simultaneously, the R values dropped and Ps went up. In the case of the rabbit rectal epithelium, a similar treatment did not elicit significant changes in the water permeability during the first 20 min following the osmotic challenge while there was a significant increase in the transepithelial resistance. After exposure to serosal hypertonicity, several morphological modifications developed in the Caco-2 cells: Localized dilations in the intercellular spaces and vacuoles in the cytoplasm appeared. Nevertheless, most cells remained in contact and no evidence of cell shrinking was observed. Simultaneously, the tight-junction structure was more or less disorganized. The filament network lost its sharpness and "omega" figures appeared, bordering the intercellular spaces. In some cases the tight-junction network was completely disrupted. In the case of the rabbit rectum the structural modifications were completely different: Serosal hypertonicity rapidly induced cell shrinking and the opening of the intercellular spaces, with no noticeable change in the tight-junction structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.

Freeze-fracture Transmission Electron Microscopy (FF/TEM) studies of systems containing self-associated structures

Freeze-fracture transmission electron microscopy (FF/TEM) is a well-established and highly-valued technique, often employed in the study of biological systems which are extremely sensitive to structural alteration (e.g., membranes and tissues). The technique relies on rapid specimen cooling to immobilize detailed microstructure, usually in a hydrated environment, prior to fracture and subsequent surface replication. As Zasadzinski and Bailey point out, though, the principle governing this technique is general and can be applied with equal success to the study of “microstructured” or “complex” fluids, i.e., fluids consisting of self-organized supramolecular structures. In this vein, FF/TEM constitutes a powerful means of characterizing the structural attributes of dispersions, emulsions, gels, and liquid crystalline assemblies at relatively high spatial resolution. Such morphological information can prove valuable in the development of the structure-viscosity relationships needed in processing. Here, we demonstrate the utility of FF/TEM in elucidating the role of self-associated structures in three different systems: a chemical reaction environment, a high-internal-phase emulsion, and a nonaqueous gel.

X-ray diffraction and freeze-fracture electron microscopy study of the cubic phase in the cetylpyridinium chloride/formamide and cetyltrimethylammonium chloride/formamide systems

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTX-ray diffraction and freeze-fracture electron microscopy study of the cubic phase in the cetylpyridinium chloride/formamide and cetyltrimethylammonium chloride/formamide systemsX. Auvray, M. Abiyaala, P. Duval, C. Petipas, I. Rico, and A. LattesCite this: Langmuir 1993, 9, 2, 444–448Publication Date (Print):February 1, 1993Publication History Published online1 May 2002Published inissue 1 February 1993https://doi.org/10.1021/la00026a014RIGHTS & PERMISSIONSArticle Views153Altmetric-Citations19LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Formation of polyester blends by a recombinant strain ofPseudomonas oleovorans: Different poly(3-hydroxyalkanoates) are stored in separate granules

WhenPseudomonas oleovorans (GPo1) is grown on sodium octanoate under ammonium limiting conditions, it is able to accumulate a copolyester consisting of medium chain length 3-hydroxyalkanoic acids (PHAm). 3-Hydroxybutyrate is only incorporated in trace amounts. WhenP. oleovorans is equipped with the PHB biosynthetic genes ofAlcaligenes eutrophus (GPo1[pVK101::PP1]), it forms a polyester containing major amounts of 3-hydroxybutyrate. The resulting polymer however is a blend of PHAm and PHB, rather than a copolymer of 3-hydroxybutyrate and medium chain length 3-hydroxyalkanoic acids [11]. To establish whether PHAm and PHB molecules are stored in the same or separate granules by this recombinantP. oleovorans strain, we studied polymer forming cells by freeze-fracture electron microscopy. This approach is possible because previous freeze-fracture electron microscopy studies on PHAm and PHB accumulating strains have shown that PHAm and PHB granules can be distinguished from each other: PHAm granules from mushroom-like structures, whereas PHB granules from needle structures during freeze-fracturing. In this paper we show that stationary phase cells of GPo1[pVK101::PP1] contained both mushroom and needle-like structures, indicating that PHAm and PHB chains were stored in separate granules. To be able to determine whether the separation of PHAm and PHB is complete, the respective granules were separated on sucrose gradients. A total cell extract of GPo1[pVK101::PP1] which was subjected to sucrose gradient centrifugation revealed two white bands of different densities: the upper band with a density of 1.05 g/mL consisted exclusively of PHAm granules, while the lower band with a density of 1.19 g/mL consisted of PHB granules only. Thus, when bacteria synthesize both PHAm and PHB, the resulting polymer chains are segregated completely and stored in separate granules.