Molecular order and mobility within membranes made from fluorocarbon/fluorocarbon and fluorocarbon/hydrocarbon double-chain ether and amide phospholipids

The order of membranes formed by various ether- or amide-connected fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon double-chain phosphocholines which derive, respectively, from glycerol or diaminopropanol or serine, has been investigated using fluorescence anisotropy of TMA-DPH, and compared to that of membranes made from fluorinated or conventional ester-connected phosphatidylcholines. In the gel phase, membrane order is almost not affected by the molecular structure of the phospholipid. By contrast, in the liquid-crystalline state, it decreases on going (i) from membranes made from the fluorocarbon/fluorocarbon phospholipids to those formed by mixed fluorocarbon/hydrocarbon phospholipids, then to conventional ones, (the rigidifying effects of the fluorinated tails, their number and length is thus becoming noticeable) or (ii) from membranes made from fluorinated diamidopropano- or diamidoserinophosphocholines to those made from ester- or ether-connected glycerophosphocholines. The ester- versus ether-connection or 1,2- versus 1,3-connection of the chains on glycerol have no detectable impact on membrane order.     

Long-term perfusion of the isolated rat liver maintenance of its functional state by use of a fluorocarbon emulsion

In order to establish a long-term perfusion system a fluorocarbon emulsion was developed and employed for the perfusion of isolated rat liver up to 20 h. Its suitability for maintaining some specific organ functions was compared with that of a commonly used red cell-containing medium. All livers perfused with the fluorocarbon medium released phosphoglucose isomerase, glutamate-oxaloacetate transaminase and glutamate dehydrogenase almost linearly at a low basal rate, glutamate dehydrogenase release beginning after 5 h perfusion. In contrast to that, a certain percentage of the livers perfused with the red cell-containing medium showed an exponential enzyme release which was over two standard deviations above the mean of the livers perfused with fluorocarbon medium, the values being 25% for phosphoglucose isomerase, 38% for glutamate-oxaloacetate transmiinase and 87% for glutamate dehydrogenase after 10 h of perfusion. In each case the exponential release of phosphoglucose isomerase signaled the functional impairment of the preparation.Thus, defining those livers as “intact” only if their phosphoglucose isomerase release was within two standard deviations of the means of the fluorocarbon-perfused livers, the following liver functions were examined in fluorocarbon-perfused and, for comparison, in “intact” cell-perfused livers during a 10-h period: Metabolite state, galactose elimination from the perfusate, induction of tyrosine aminotransferase by dexamethasone, and gluconeogenesis from lactate and bile production. It was found that the fluorocarbon medium provided at least the same or an even better hepatic function than did the red cell-containing medium. However, while in red cell-perfused livers functional impairment always occurred at various percentages under the conditions mentioned above, this was never observed with the fluorocarbon medium.Electron microscopic examination of the livers perfused with the fluorocarbon medium showed no disturbance of the mitochondrial matrix and cristae after a 10 h perfusion. While within a large number of liver cells the ergastoplasm was seen in normal appearance, in other liver cells the cisternae of rough endoplasmic reticulum were vacuolated.Some important physicochemical data of the fluorocarbon medium such as O₂ capacity, viscosity and particle size are reported, and the technique and the problems of its preparation are described. The advantages of the fluorocarbon medium for long as well as short term perfusion experiments are discussed.     

Blood fluorocarbon levels following exposure to household aerosols

Measurement of blood fluorocarbon levels was carried out in subjects exposed to a number of household aerosol products containing fluorocarbon gases as propellants. All aerosols were used according to manufacturers'' recommendations. The general purpose of the study was to determine whether with normal routine exposure to a wide variety of household aerosol products fluorocarbon could be found in the blood and if so, to check the potential toxicity of the levels reached. Daily exposure to aerosols according to the study regimen lasted four consecutive weeks; this period was preceded and followed by two weeks of abstention from exposure. At the end of each of the three periods a medical examination, including evaluation of cardiac function, respiratory function studies, and hematological and functional clinical biochemical tests was performed. All tests and assessments failed to show any indication of toxicity. No trace of fluorocarbon was found in any of the blood sampled at intervals during the study.     

Synthesis and preliminary biochemical assessment of ethyl-terminated perfluoroalkylamine oxide surfactants

The synthesis and usefulness in membrane biochemistry of a new class of surfactants have been investigated. 1-Ethyl-2-dimethylamine oxide polar heads were grafted onto a hydrocarbon, a fluorocarbon or an ethyl-capped fluorocarbon hydrophobic tail. The ability of the resulting surfactants to extract and/or to stabilize in aqueous solution a test membrane protein, cytochrome b(6)f, was evaluated. While it is not a detergent, the hemifluorinated derivative efficiently kept purified cytochrome b(6)f soluble, native and functional. The data suggest that alkyl-capped fluorocarbon surfactants provide an interesting alternative to classical detergents for handling membrane proteins in aqueous solutions under non-dissociating conditions.     

Fluorinated and hemifluorinated surfactants derived from maltose: synthesis and application to handling membrane proteins in aqueous solution

Two novel fluorinated surfactants have been obtained by grafting by radical reaction either a fluorocarbon or an ethyl end-capped fluorocarbon chain onto the double bond of beta-D-allyl maltose. The two compounds thus obtained form polydisperse aggregates in water. They can keep membrane proteins water-soluble, but the protein/surfactant complexes are polydisperse, which affects neither the native state nor the stability of the proteins.     

Design, synthesis and preliminary biological evaluations of novel amphiphilic drug carriers

The synthesis of a new fluorocarbon amphiphilic drug carrier is described. A polyfunctional amino acid endowed with a fluorocarbon chain and a sugar moiety providing the amphiphilic character constitutes the central element of this structure. A (14)C-radiolabelled acetyl group was grafted onto the third function and the bioavailability of this molecule was specified in mice after IV administration. This amphiphilic drug carrier exhibits a rapid and homogeneous distribution to the whole tissues and slow elimination half-lives (higher than one day) through a biliary excretion without any toxicity (no measured DL 50 for concentrations up to 500 mg/kg).     

Microvascular gas embolization clearance following perfluorocarbon administration.

Effective treatment of vascular gas embolism may be possible with emulsified fluorocarbon compounds. We tested the hypothesis that a fluorocarbon emulsion delivered before gas embolization would enhance bubble motion through the vasculature, favoring more rapid clearance. Air microbubbles were injected into the rat cremaster microcirculation in six groups of rats receiving Perftoran, an emulsified fluorocarbon, or saline immediately before, 2 h before, or after bubble injection. Embolism dimensions and dynamics were observed by using intravital microscopy. Surface area at lodging was equal between groups. Bubbles having smaller volume embolized smaller diameter vessels in the Perftoran pretreatment groups. A higher incidence of bubble dislodgement and larger distal displacement occurred in these two groups, with a 36% decrease in the time to bubble clearance and restoration of blood flow. Intravascular emulsified fluorocarbon administration before gas embolization affected initial bubble conformation, increased bubble dislodgement, and resulted in bubble displacement further into the periphery of the microcirculation. These dynamic events did not occur if embolization preceded fluorocarbon administration.     

Formation of biologically active substances during cholesterol oxidation on the surface of fluorocarbon emulsions

Oxidative modification of cholesterol on the surface of fluorocarbon emulsions was studied. The oxidation yielded one primary product--7-peroxycholesterol. It was shown that the obtained cholesterol C7 derivatives possess a high biological activity. It was concluded that the possibility of oxidative modification of plasma substances on the surface of fluorocarbon emulsion particles with the formation of highly active compounds must be taken into account when using the fluorocarbon particles in medicine.     

Hormonal control of cardiac lipolysis by glyco(geno)lysis

The importance of the glucose/fatty acid cycle in the control of cardiac lipolysis is emphasized by the following observations. Addition of the glycogen debranching inhibitor deoxynojirimycin or an O2-vehicle, fluorocarbon F-43, to media perfusing paced, lipid-enriched, Langendorff hearts lower cardiac lactate and glycerol 3-phosphate levels together with inhibition of glucagon-stimulated glycerol (and lactate) release. The absence of fluorocarbon during perfusion of 5 Hz paced langendorff hearts probably results in limited tissue oxygenation, resulting in glycogenolysis and lipolysis. The results indicate hormonal control of cardiac lipolysis by glyco(geno)lysis.     

Culture of human adult endothelial cells on liquid-liquid interfaces: A new approach to the study of cell-matrix interactions

Summary Human adult endothelial cells (ECs) were cultured on liquid-liquid interface formed when aqueous culture medium is overlaid onto a fluorocarbon solvent. When ECs were seeded on untreated interfaces, some cells seemed to attach but they did not spread or grow. In contrast, when ECs were seeded on interfaces pretreated with such proteins as collagen type IV (COL), laminin (LN), fibronectin (FN), and fibrinogen (FG) the cells spread and proliferated until they formed confluent monolayers. Proteins such as bovine serum albumin (BSA) or gelatin (GN) were not as effective in providing surfaces for vigorous growth. Cells grown on fluorocarbon interfaces expressed specialized characteristics exhibited by endothelial cells grown under the usual culture conditions; they grew in a cobblestone monolayer, stained positively for Factor VIII-related antigen, and produced angiotensin-converting enzyme. The growth rate of ECs was the same whether they were cultured on treated fluorocarbon interfaces or on the usual tissue culture plastic surfaces. Using this culture system, the interactions of ECs with various adhesive proteins used as substrata was examined. ECs were observed to attach readily to the interfaces coated with GN, COL, LN, FN, and FG, but poorly to those coated with BSA. All the substrates tested, with the exception of BSA, promoted EC growth on fluorocarbon interfaces; ECs tended to grow more rapidly on COL- or FG-coated interfaces than on LN-, FN-, or GN-coated interfaces. This work was supported in part by grants from the National Institutes of Health (R01-HL-34153 and P01-AG-04861).     

Cholesterol oxidation on fluorocarbon emulsion surface leads to the formation of 7-peroxycholesterol

Formation of biologically active oxidized derivatives of cholesterol as a result of its oxidation on the surface of fluorocarbon emulsions was studied. A single product of cholesterol oxidation, 7-peroxycholesterol, was found. It was shown that 7-peroxycholesterol and its derivative 7-keto-cholesterol inhibit the rosette formation between human T-lymphocytes and sheep erythrocytes. These substances exert a strong cytostatic action on the growth of procaryotic and eucaryotic cell cultures. Thus, oxidative modification of blood plasma components on the surface of fluorocarbon emulsion particles with the formation of highly active compounds must be taken into account when using the fluorocarbon emulsions in medicine.     

Adhesion and proliferation of fibroblasts on RF plasma-deposited nanostructured fluorocarbon coatings: evidence of FAK activation

We used combined plasma-deposition process to deposit smooth and nanostructured fluorocarbon coatings on polyethylenethereftalate (PET) substrates, to obtain surfaces with identical chemical composition and different roughness, and investigate the effect of surface nanostructures on adhesion and proliferation of 3T3 Swiss Albino Mouse fibroblasts. Untreated PET and polystyrene (PS) were used as controls for cell culture. We have found that the statistically significant increase of cell proliferation rate and FAK (a nonreceptor tyrosine kinase) activation detected on ROUGH fluorocarbon surfaces is due to the presence of nanostructures. Changes in cytoskeletal organization and phospho FAK (tyr 397) localization were evident after 60 min on cells adhering to ROUGH surfaces. This change was characterized by the formation of actin stress fibers along lamellar membrane protrusion instead of usual focal contacts. Also the morphology of the adhering fibroblasts (60 min) adhering on ROUGH surfaces was found quite different compared to cells adhering on smooth ones.     

The ability of oxygenated fluorocarbon solution to minimize ischemic skeletal muscle injury

The amputated canine limb was used to create a devascularized skeletal-muscle-containing model. The extremity was perfused with one of several solutions or merely cooled as a preservative measure during the devascularized period and then replanted. Perfusion of the limb with oxygenated fluorocarbon solution minimized edema formation and leakage of skeletal muscle enzymes into the serum following revascularization as compared to the other perfusates or to mere cooling. Histopathologic changes within the revascularized muscle were also minimized by the oxygenated fluorocarbon perfusion. It is probable that these findings will correlate with improved function of replanted extremities or free muscle flaps. The applicability of these findings to human extremity replantation and free-flap transfer is postulated. However, long-term human studies will be necessary to assess the function of extremities and flaps perfused with this solution prior to its general use in extremity replantation and free-flap transfer.     

The differential response of the skin in young and old rats to a combination of X-rays and 'wet' or 'dry' hyperthermia

The left hind feet of groups of female rats aged 7, 14 and 52 weeks were irradiated at three dose levels of X-rays (20, 25 or 30 Gy). Hyperthermia (42.5 degrees C for 1 h) was carried out immediately following irradiation using either 'wet' or 'dry' heat, achieved by immersion in either water or fluorocarbon liquid. The results demonstrated that 'wet' heat produced a consistently greater enhancement of the irradiation damage than 'dry' heat. The thermal enhancement ratio for irradiation plus 'wet' heat was approximately 1.5 and for irradiation plus 'dry' heat it was in the range 1.17 to 1.39. Immersion of the feet in fluorocarbon liquid at 37 degrees C did not significantly modify the irradiation response of the skin. The lower thermal enhancement ratios obtained using immersion in fluorocarbon liquid at 42.5 degrees C are close to those obtained in large animal studies and also similar to the limited amount of data from clinical studies where microwave or ultrasound heating techniques were used. It has been demonstrated that there are large age-related differences in the response of the rat foot skin to irradiation alone. It has also been shown in the present study, using rats of the same age, that the response to irradiation plus hyperthermia was less age dependent. This finding may reflect the differing methods by which damage occurs in tissue after irradiation or hyperthermia.     

Phase behavior of fluorocarbon and hydrocarbon double-chain hydroxylated and galactosylated amphiphiles and bolaamphiphiles. Long-term shelf-stability of their liposomes.

This paper describes the morphological characterization, by freeze-fracture electron microscopy, and the thermotropic phase behavior, by differential scanning calorimetry and/or X-ray scattering, of aqueous dispersions of various hydroxylated and galactosylated double-chain amphiphiles and bolaamphiphiles, several of them containing one or two hydrophobic fluorocarbon chains. Colloidal systems are observed in water with the hydroxylated hydrocarbon or fluorocarbon bolaamphiphiles only when they are dispersed with a co-amphiphile such as rac-1,2-dimyristoylphosphatidylcholine (DMPC) or rac-1,2-distearoylphosphatidylcholine (DSPC). Liposomes are formed providing the relative content of bolaamphiphiles does not exceed 20% mol. Most of these liposomes can be thermally sterilized and stored at room temperature for several months without any significant modification of their size and size distribution. The hydrocarbon galactosylated bolaamphiphile HO[C24][C12]Gal forms in water a lamellar phase (the gel to liquid-crystal phase transition is complete at 45 degrees C) and a Im3m cubic phase above 47 degrees C. The fluorocarbon HO[C24][F6C5]Gal analog displays a more complex and metastable phase behavior. The fluorinated non-bolaform galactosylated [F8C7][C16]AEGal and SerGal amphiphiles form lamellar phases in water. Low amounts (10% molar ratio) of the HO[C24][F6C5]Gal or HO[C24][C12]Gal bolaamphiphiles or of the single-headed [F8C7][C16]AEGal improve substantially the shelf-stability of reference phospholipon/cholesterol 2/1 liposomes. These liposomes when co-formulated with a single-headed amphiphile from the SerGal series are by far less stable.     

Preservation of Tracheal Mucus by Nonaqueous Fixative

Two nonaqueous fixatives, composed of fluorocarbon solvents with dissolved osmium tetroxide, were used to determine the feasibility of preserving the mucous coat in bovine and rat trachea for light and electron microscopy. Aqueous fixatives, while providing excellent cytological preservation, wash away the mucous lining, precluding ultrastructural analysis. Inclusion of ruthenium red or alcian blue within aqueous fixative improved retention of mucus, but provided incomplete, patchy results. Fixation with nonaqueous fluorocarbon solvent and dissolved osmium tetroxide preserved a continuous mucous epiphase layer above a clear hypophase layer. Subcomponents of the mucus included an electron dense surface layer, interrupted patches of mucus above the surface layer and electron dense membrane-like material within the mucus. This method of fixation will preserve mucus for light, scanning and transmission electron microscopy, using either intratracheal or immersion methods of fixation. The latter would enable use of materials from large animal models, autopsy or an abattoir.     

Kinetics of the reactions involving CF2 and CF in a pure tetrafluoromethane plasma: II. Production and loss of CF2 and CF in the processes of fluorocarbon polymerization

Mechanisms for the production and loss of CF₂ and CF radicals in a glow discharge in pure CF₄ are investigated by the time-resolved laser-induced fluorescence method. The fluorocarbon polymerization processes are shown to contribute significantly to the production of radicals both in the plasma volume and on the surface of the discharge tube. The effective frequencies of both the volume and surface processes of radical production and loss are determined. An analysis of these frequencies allowed us to study the polymerization mechanism in a CF₄ plasma at a high relative concentration of F atoms and low ion energy. It is shown that, at elevated pressures, when the density of C_xF_y polymer particles in the plasma volume becomes comparable with the density of simple fluorocarbon radicals, the electron-impact dissociation of these particles is the main channel for the production of CF₂ and CF radicals. Another source of CF₂ and CF radicals is related to the reactions of C_nF_2m+1 unsaturated fluorocarbon particles both in the plasma volume and on the surface of a fluorocarbon film arising on the discharge tube wall. The C_xF_y fluorocarbon polymer particles form both in the discharge volume and on the fluorocarbon filmsurface also in the course of the film destruction. At lowered pressures, the main channel for the production of CF₂ and CF is the direct electron-impact dissociation of CF₄ molecules, whereas the loss of these radicals at the tube wall is the main loss channel. The probabilities of the heterogeneous losses of CF₂ and CF radicals on the heavily fluorinated surface of the fluorocarbon film at low ion energies are determined. Under these conditions, the surface recombination of the Fch chemisorbed fluorine atoms and CF _x ^ph physisorbed radicals with the production of an activated complex is shown to be the most probable mechanism for the heterogeneous losses of CF₂ and CF. The approximate activation energies for the production of F_ch · CF ₂ ^ph and F_ch · CF^ph surface complexes are found to be 750±70 K and 1030±100 K, respectively.     

Antibody immobilization onto glow discharge treated polymers.

Previous studies have shown that certain glow discharge treated polymers strongly retain adsorbed albumin and fibrinogen. On the basis of this phenomenon, we have investigated the possibility of immobilizing antibodies on glow discharge treated surfaces for diagnostic immunoassay applications. As a model for antibody immobilization, bovine IgG was immobilized on the following polymers: polyethylene (PE), tetrafluoroethylene glow discharge treated PE (TFE/PE), poly(ethylene terephthalate) (PET), TFE/PET, poly(tetrafluoroethylene) (PTFE), ethylene glow discharge treated PET (E/PET) and hexamethyldisiloxane glow discharge treated PET (HMDS/PET). IgG was radiolabeled with 125I and immobilized by either of the following two methods: (a) physical adsorption of IgG on untreated and glow discharge treated polymers or (b) physical adsorption of albumin followed by chemical coupling of IgG to albumin by glutaraldehyde. IgG concentration as well as adsorption times were varied in order both to optimize the immobilization conditions and to investigate the adsorption and retention mechanisms. To evaluate the efficiency of the immobilization techniques, blood plasma, Tween-20, and sodium dodecyl sulfate (SDS) were used to elute the adsorbed IgG layer. We found that IgG was successfully immobilized on the fluorocarbon glow discharge treated surfaces by using either the physical adsorption or the glutaraldehyde coupling method, although the former is more efficient than the latter method.     

Structures of trichovirins II, peptaibol antibiotics from the mold Trichoderma viride NRRL 5243.

From the culture broth of the mold Trichoderma viride NRRL 5243 a mixture of polypeptides, named trichovirins (TV), could be isolated and purified by chromatography on XAD-2 adsorber resin and Sephadex LH-20 gel. Chromatography on silica gel using chloroform/methanol 8:2 as eluent provided a mixture of peptides named TV I. Subsequent elution with chloroform/methanol 1:1 yielded a second group of peptides named TV II. That group could be separated into individual components by repetitive HPLC on an octadecylsilyl and a fluorocarbon stationary phase. The sequences of 12 peptides of TV II could be determined by electrospray ionization tandem mass spectrometry of isolated peptides and gas chromatography-mass spectrometry of methanolysates. The N-termini of the 18-mer peptides are acetylated and the C-termini consist of leucinol. Owing to the presence of alpha-aminoisobutyric acid (Aib) residues and the bactericidal and hemolytic activity, the peptides belong to the family of peptaibol antibiotics.     

Use of macroporous polypropylene filter to allow identification of bacteria by PCR in human fecal samples

The detection of pathogenic bacteria directly in human fecal specimens by PCR, requires removal of PCR-inhibitory substances. To investigate whether five different macroporous filters (polypropylene, nylon, polyester, polyethylene, fluorocarbon) could retain polysaccharides, major PCR inhibitors, an in vitro model and human fecal samples were used. The in vitro model consisted of Xanthum gum solutions (3 mg/ml PBS), a bacterial polysaccharide, to which Helicobacter pylori cells were added. Fecal samples from healthy volunteers were spiked with H. pylori and Mycobacterium paratuberculosis cells. Polysaccharide concentrations were significantly reduced only by the polypropylene but not by the other filters. Accordingly, both Xanthum gum solutions and spiked fecal specimens became PCR positive only after filtration with the polypropylene filter. We conclude that this filter can be used to prepare a bacterial DNA template suitable for PCR analysis from human feces.