Plastid transformants of tomato selected using mutations affecting ribosome structure.

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.

Study of the toxicity of a new lipid complex formulation of amphotericin B.

OBJECTIVES: The aim of this study was to evaluate the toxicity of a new lipid complex formulation of amphotericin B (LC-AmB) produced by a simple process. METHODS: Toxicity was evaluated after daily administration for 21 consecutive days in female CD1 mice. Doses of LC-AmB up to 20 mg/kg were used, and compared with Fungizone at 0.5 mg/kg and Abelcet at 10 mg/kg. Acute toxicity after a single bolus injection was also determined, as well as the haemolytic activity and toxicity to mouse macrophages in vitro. RESULTS: LC-AmB reduced both the haemolytic activity of amphotericin B and its toxicity towards mouse peritoneal macrophages. Its acute toxicity (LD50 > 200 mg/kg in CD1 mice) was similar to that in the literature for the least toxic lipid formulations of amphotericin B. The relative liver weight increased slightly in mice treated daily with a dose of 20 mg/kg LC-AmB, as did the kidney weight in this group and the group treated with Fungizone. There was also a dose-dependent decrease in the haematocrit with all formulations. All treatments caused significant increases in transaminase levels. Total hepatic CYP 450 was slightly but not significantly increased in the groups treated with 20 mg/kg LC-AmB, Abelcet and Fungizone. However, expression of some isoforms of CYP 450 was reduced, the most marked being the hepatic CYP 3A1 after treatment with 20 mg/kg LC-AmB, Abelcet and Fungizone. The effects on hepatic function are probably related to accumulation in organs rich in phagocytic cells. CONCLUSION: LC-AmB did not induce any new toxicity compared with Abelcet and Fungizone.

Intralipid 10%: physicochemical characterization.

OBJECTIVES: Parenteral fat emulsions contain two populations of particles: artificial chylomicrons rich in triacylglycerols (TAG), and liposomes (bilayer of phospholipids [PL] enveloping an aqueous phase). Centrifugation permits isolating the liposomes in the infranatant called mesophase. The aim of the present work was to better characterize this mesophase chemically and to view the particles it contains by electron microscopy. METHODS: Electron microscopy (Philips 410) was performed after cryofracture on native 10% Intralipid, mesophase (centrifugation for 1 h at 27 000 g), and a liposome-enriched fraction (ring of density 1.010-1.030 g/l obtained after centrifuging mesophase in a KBr density gradient at 100 000 g for 24 h). The TAG and protein content of the mesophase was analyzed and the proteins partially characterized by immunodetection (Western-blot). RESULTS: This electron microscope study of 10% Intralipid gives evidence for the coexistence of artificial chylomicrons (mean diameter, 260 nm) and liposomes (43 nm), the latter being smaller than expected and containing 8% w/w TAG after purification. The solubilization of TAG in PL bilayers (reported to be < or = 3.1% w/w) might have been increased in parenteral emulsions by the manufacturing process or/and the high TAG/PL ratio. Minute amounts of proteins have also been detected and partially characterized using a specific antibody raised against the human 7 kDa Anionic Polypeptide Factor (APF), known to strongly interact with PL in bile. CONCLUSIONS: This work has shown that the size (mean diameter, 43 nm) of the liposomes present in 10% Intralipid is smaller than that usually assumed. Traces of hydrophobic proteins in the emulsion may account for certain allergic reactions sometimes observed in infused patients.

Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

Polyisobutylcyanoacrylate nanocapsules containing an aqueous core for the delivery of oligonucleotides.

Antisense oligonucleotides (ODNs) with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using ODNs as active molecules, the development of nanocapsules containing ODNs in their aqueous core was considered. Nanocapsules were prepared by interfacial polymerization of isobutylcyanoacrylate (IBCA) in a W/O emulsion. After ultracentrifugation and re-suspension in water, the nanocapsules displayed a size of 350+/-100 nm. Oligonucleotide loading did not significantly influence the zeta potential, suggesting that they were located within the core of the nanocapsules. Fluorescence quenching assays confirmed this localization. When encapsulated in the nanocapsules and incubated in the presence of serum, the ODNs were efficiently protected from degradation by nucleases, whereas ODNs adsorbed onto nanospheres were less efficiently protected. This paper describes, for the first time, a nanotechnology able to encapsulate ODNs, rather than adsorbing them at the surface of a solid support. Such a formulation has great potential for oligonucleotide delivery.

Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotides.

PURPOSE: The goal of the present paper was to encapsulate oligonucleotides in a new particulate carrier in order to protect them from enzymatic degradation. METHODS: Nanocapsules with an aqueous core containing oligonucleotides were prepared by interfacial polymerization of isobutylcyanoacrylate in a W/O emulsion. Ultracentrifugation and re-suspension in water yielded a dispersion of these containing an aqueous core nanocapsules. Zeta potential measurements and quenching of fluorescence of fluorescein-bounded oligonucleotides were used to study the localization of the oligonucleotides. Oligonucleotide degradation studies were carried out in fetal calf serum. RESULTS: Polydisperse nanocapsules of size ranging from 20 to 400 nm were obtained. Oligonucleotide loading did not significantly influence the zeta potential, suggesting they were located within the core of the nanocapsules. Fluorescence quenching assays confirmed this localization. When encapsulated in the nanocapsules and incubated in the presence of serum, the oligonucleotides were efficiently protected from degradation by nucleases, whereas oligonucleotides adsorbed onto nanospheres were protected less efficiently. CONCLUSIONS: This paper describes, for the first time, a nanotechnology able to encapsulate oligonucleotides rather than adsorbing them at the surface of a solid support. Such a formulation has great potential for oligonucleotide delivery.

Myosin thick filaments from adult rabbit skeletal muscles.

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).

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.

Visualization of in vitro protein-rejecting properties of PEGylated stealth polycyanoacrylate nanoparticles.

The in vitro protein-rejecting properties of PEG-coated polyalkylcyanoacrylate (PACA) nanoparticles were for the first time visualized after freeze-fracture of the nanoparticles pre-incubated with fibrinogen as a model blood protein. The reduced protein association to the nanoparticles was evidenced also by two-dimensional PAGE after incubation of the nanoparticles with human plasma. In vivo experiments showed the 'stealth' long-circulating properties of the PEGylated nanoparticles after intravenous administration to mice. Thus, the images obtained after nanoparticle-protein incubation were predictive of the behavior observed in vivo. In conclusion, freeze-fracture analysis represents a novel and original qualitative approach to investigate the interactions between proteins and particulate systems.

Self-assembly of chlorophenols in water.

In saturated solutions of some di- and trichlorophenols, structures with complex morphologies, consisting of thin, transparent sheets often coiling into helices and ultimately twisting into filaments, were observed under the optical microscope. Freeze-fracture electron microscopy, x-ray diffraction, phase diagrams, and molecular modeling were performed to elucidate the observed phenomena. Here, we present evidence that the chlorophenols studied, when interacting with water, self-assemble into bilayers. The fact that some chlorophenols form the same supramolecular structures as those described previously for structurally nonrelated surfactants sheds light on the mechanisms of self-assembly.

A polysorbate-based non-ionic surfactant can modulate loading and release of beta-lactoglobulin entrapped in multiphase poly(DL-lactide-co-glycolide) microspheres.

PURPOSE: The goal of the present paper was to investigate the role of a surfactant, Tween 20, in the modulation of the entrapment and release of beta-lactoglobulin (BLG) from poly (DL-lactide-co-glycolide) microspheres. METHODS: Poly(DL-lactide-co-glycolide) microspheres containing BLG were prepared by a water-in-oil-in-water emulsion solvent procedure. Tween 20 was used as a surfactant in the internal aqueous phase of the primary emulsion. BLG entrapment efficiency and burst release were determined. Displacement of BLG from microsphere surface was followed by confocal microscopy observations and zeta potential measurements, whereas morphological changes were observed by freeze-fracture electron microscopy. RESULTS: Tween 20 was shown to increase 2.8 fold the encapsulation efficiency of BLG without any modification of the stability of the first emulsion and the viscosity of the internal aqueous phase. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface or very close to the surface as shown by confocal microscopy and zeta potential measurements. Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communications with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease of the burst release. CONCLUSIONS: These results constitute a step forward in the improvement of existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method.

Crystallographic analysis of freeze-fractured three-dimensionally ordered specimens.

We describe here an original approach for solving the structure of three-dimensionally ordered specimens at low and medium resolutions. It combines freeze-fracture electron microscopy and quantitative image processing and has been first successfully applied to the crystallographic study of different lipid-containing cubic phases. The structure preservation during cryofixation is controlled by recording X-ray diffraction before and after freezing. Well frozen cubic phases show fracture planes which look like well defined cleavage planes of 3-D crystals. These fracture planes (domains) reveal a mosaic of 2D ordered sub-domains which are geometrically related to each other by simple crystallographic operations. The symmetry properties of the images mirror faithfully the symmetry of the space groups. The shifts and rotations observed between adjacent sub-domains are related to this symmetry. Different cubic phases display different fracture behavior, highly characteristic for a given space group. Interpretation of the averaged images of different domains in terms of molecular structure is done by the comparison of the averaged periodic motifs either with the corresponding sections of the electron density map (from X-ray diffraction data) or with the corresponding sections of a 3-D-space filling model. We show here that the same procedure may be applied to other three-dimensionally ordered specimens such as 3-D crystals of membrane proteins or of other proteins, including naturally occurring protein crystals of some secretory organelles. Finally, the same approach could also provide a powerful tool for the study of membrane protein crystallogenesis, particularly for the formation of 3-D crystals.

Liposomes dispersed within a thermosensitive gel: a new dosage form for ocular delivery of oligonucleotides.

PURPOSE: The main goal of this study was to develop an ocular controlled release formulation of a model oligonucleotide (pdT16), contained within liposomes dispersed within a thermosensitive gel composed by poloxamer 407. METHODS: The influence of the poloxamer concentration 2% or 27% on the stability of the liposomes (PC: CHOL and PC: CHOL: PEG-DSPE) was investigated. The in vitro release profiles of pdT16 from various poloxamer formulations (free pdT16 dispersed within 20% and 27% poloxamer gels, pdT16 encapsulated within liposomes dispersed within 20% and 27% poloxamer gels) were realized using a membrane-free release model. RESULTS: The dispersion of liposomes within a dilute 2% poloxamer solution resulted in a great leakage of pdT16 from liposomes. However, the destabilization effect of poloxamer was reduced when higher concentration (27%) was used. Poloxamer dissolution was found to control the release process of pdT16, whereas the dispersion of liposomes within 27% poloxamer gel was shown to slow down the diffusion of pdT16 out from the gel. CONCLUSIONS: The dispersion of liposomes within a 27% poloxamer gel presented an interesting system to control the release of a model oligonucleotide compare to a simple gel.

Pegylated nanoparticles from a novel methoxypolyethylene glycol cyanoacrylate-hexadecyl cyanoacrylate amphiphilic copolymer.

PURPOSE: The aim of this work was to develop PEGylated poly(alkylcyanoacrylate) nanoparticles from a novel methoxypolyethyleneglycol cyanoacrylate-co-hexadecyl cyanoacrylate copolymer. METHODS: PEGylated and non-PEGylated nanoparticles were formed by nanoprecipitation or by emulsion/solvent evaporation. Nanoparticles size, zeta potential and surface hydrophobicity were investigated. Surface chemical composition was determined by X-ray photoelectron spectroscopy. Nanoparticle morphology was investigated by transmission electron microscopy after freeze-fracture. Nanoparticles cytotoxicity was assayed in vitro, onto mouse peritoneal macrophages. Cell viability was determined through cell mitochondrial activity, by a tetrazolium-based colorimetric method (MTT test). Finally, the degradation of PEGylated and non-PEGylated poly(hexadecyl cyanoacrylate) nanoparticles was followed spectrophotometrically during incubation of nanoparticles in fetal calf serum. RESULTS: Monodisperse nanoparticles with a mean diameter ranging between 100 and 200 nm were obtained using nanoprecipitation or emulsion/solvent evaporation as preparation procedures. A complete physico-chemical characterization, including surface chemical analysis, allowed to confirm the formation of PEG-coated nanoparticles. The PEGylation of the cyanoacrylate polymer showed reduced cytotoxicity towards mouse peritoneal macrophages. Furthermore, the presence of the PEG segment increased the degradability of the poly(hexadecyl cyanoacrylate) polymer in presence of calf serum. CONCLUSIONS: We succeeded to prepare PEGylated nanoparticles from a novel poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecyl cyanoacrylate) by two different techniques. Physico-chemical characterization showed the formation of a PEG coating layer. Low cytotoxicity and enhanced degradation were also shown.

Phase behavior of fluorocarbon di-O-alkyl-glycerophosphocholines and glycerophosphoethanolamines and long-term shelf stability of fluorinated liposomes.

This paper is devoted to the morphological characterization, by freeze-fracture electron microscopy, and to the thermotropic phase behavior, by differential scanning calorimetry and X-ray diffraction of the aqueous dispersions of various fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon 1,2- or 1,3-di-O-alkyl-glycerophosphocholines (PC) and 1,2-di-O-alkyl-glycerophosphoethanolamines (PE). The fluorinated PCs form classical lamellar phases and liposomes while an interdigitated lamellar phase has been evidenced for a hydrocarbon 1,3-analog. The fluorinated PEs display a lamellar to hexagonal phase transition which occurs almost simultaneously with the gel-to-fluid lamellar phase transition. The impact of each of the structural features [ether vs ester chemical junction connecting the hydrophobic chains on glycerol, their position (1,2- vs 1,3 isomers), number and length of the perfluoroalkylated chains, length of the fluorinated tail and hydrocarbon spacer, PC vs PE polar head] of the fluorinated phospholipids on the phase transition thermodynamic parameters (Tc, delta H, delta S) is discussed. Most of the liposomes formed from the fluorinated ether-PCs display a remarkable long-term shelf stability: they can be thermally sterilized and stored at room temperature for several months without any significant modification of their size and size distribution.

Effect of physical constraints on the mechanisms of membrane fusion: bolaform lipid vesicles as model systems.

Bolaform lipid vesicles were used to study the effect of physical constraints on membrane fusion. In these vesicles the membrane is organized in a single monolayer, because of the presence of covalent bonds in its middle plane. Therefore, the formation of fusion intermediates is subject to higher energy barriers and greater geometrical constraints than is usual in bilayer membranes. Bolaform lipids were extracted from the thermophilic archaeon Sulfolobus solfataricus. These lipids can be divided into two classes, the monosubstituted molecules, in which one of the polar heads is glycerol, and the bisubstituted molecules, endowed with two complex polar heads. The fusion process in vesicles composed of different mixtures of monosubstituted/bisubstituted molecules was studied by means of fluorescence techniques. Ca2+ or poly(ethylene glycol) was employed as a fusogenic agent. We found that fusion of such constrained membranes is still possible, provided that molecules able to mediate a structural rearrangement of the membrane are present. This condition is fulfilled by monosubstituted molecules, which are able to partition the glycerol headgroup in the apolar moiety. In addition, the presence of traces (approximately 5%) of the monopolar compound diphytanylglycerol is an important factor for fusion to occur. On the contrary, vesicles formed by bisubstituted molecules are unable to fuse, irrespective of the fusogen employed.

[Computed and magnetic resonance tomography in the differential diagnosis of osteoarticular tuberculosis]. / Komp'iuternaia i magnitno-rezonansnaia tomografiia v differentsial'noi diagnostike kostno-sustavnogo tuberkuleza.

Due to their high resolving capacity, computerized and magnetic resonance tomographic examinations allow early detection of tuberculosis lesions in vertebral column, large joints and their surrounding soft tissues. They also widen the possibilities for differential diagnosis, help to individualize the choice of adequate treatment, improve the outcomes of this treatment and decrease the period of patients' intrahospital stay.

Topological properties of two cubic phases of a phospholipid:cholesterol:diacylglycerol aqueous system and their possible implications in the phospholipase C-induced liposome fusion.

Water dispersions of phospholipid:cholesterol:diacylglycerol may, under certain conditions, originate either the lipid- and water-permeable Q224 cubic phase, or the lipid-permeable but water-impermeable Q227 cubic phase. These results are discussed within the framework of the phospholipase C-induced fusion of liposomes [Nieva et al. (1993) Biochemistry 32, 1054]. It is suggested that the cubic phases Q224 and Q227 represent two classes of lipid organization, one promoting, the other hindering the mixing of aqueous contents that is characteristic of membrane fusion. In this context, inverted micelles appear to be the end point of the fusion process, rather than fusion intermediates.

A conformation transition of lung surfactant lipids probably involved in respiration.

X-ray scattering and freeze-fracture electron microscopy of a lung surfactant extract show the existence of a complex lamellar phase, L gamma, over a wide range of concentrations and temperatures. This lamellar phase, which consists of two bilayer motifs comprised of monolayers with stiff chains alternating with monolayers with disordered chains, allows us to propose a structural model of a collapse phase at the alveolar pulmonary interface. This model accounts for the increase in surface pressure during the compression as well as the easy respreading upon expansion of the interface during the respiratory cycle.