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The water-soluble antineoplastic agent 5-fluoro-2'-deoxyuridine (FUdR) was encapsulated in the water phase of liposomes of different lipid compositions. The retention of this drug upon storage and during contact with plasma was assessed. It was found that, upon refrigeration, diffusion of FUdR across the liposome bilayer was considerably faster when the drug was encapsulated in fluid-type liposomes (egg PC/PS/CHOL) than in solid-type liposomes (DSPC/DPPG/CHOL). With either composition, leakage of the drug from the liposomes was accelerated upon contact with plasma. To achieve improved liposomal retention of the drug, FUdR was converted to a lipophilic prodrug by esterifying the free hydroxyl groups in the deoxyribose moiety with fatty acids of different chain lengths. Thus FUdR-dipalmitate (C-16) and FUdR-dioctanoate (C-8) were synthesized and incorporated in liposomes. The dipalmitoyl derivative could be incorporated upto 13 mol% in solid-type liposomes but to only 2 mol% in fluid-type liposomes. Freeze-fracture electron microscopy revealed no major differences between control liposomes and those containing the prodrug. FUdR-dipalmitate was found to be firmly associated with the liposomal bilayer in both liposome-types: no exchange of the pro-drug with blood constituents or hydrolysis by serum esterases could be registered when the liposomes were incubated with serum. On the other hand, liposome-incorporated FUdR-dioctanoate was found to be readily extracted from the liposomes by serum components (predominantly albumin) and was found to be degraded rapidly by serum esterase activity. The antitumor activity of FUdR-prodrugs was determined using C26 colon adenocarcinoma cells. This cell line was found to be highly sensitive to FUdR. Liposomal FUdR-dioctanoate inhibited cell growth in the same concentration range as unesterified FUdR. FUdR-dipalmitate, however, was more than two orders of magnitude less potent in inhibiting cell proliferation. Its antiproliferative activity was dependent on the liposome-type used: when incorporated in fluid-type liposomes, antiproliferative activity of FUdR-dipalmitate was several-fold higher than in solid-type liposomes. The difference in antitumor activity between FUdR-dipalmitate and FUdR-dioctanoate and between FUdR-dipalmitate in the fluid- and solid-type liposomes could be explained by differences in the rate of hydrolysis of the prodrugs to FUdR by esterase activity in the tumor cells or in the growth medium.

Rhoptries are organelles that have important, complex roles in Apicomplexa biology. During Toxoplasma gondii infection, these organelles take part in several essential and complex processes that include host cell entry and parasite development. Using different electron microscopy techniques, we characterized the fine morphology of the rhoptries of two of the most important life stages of T. gondii: the tachyzoite and the bradyzoite forms. The observed tachyzoite and bradyzoite rhoptries had delimited regions characterized by a dark and electron-dense neck, an amorphous and less electron-dense bulb, and a region of intermediate electron density, which connects the bulb to the neck. Metal replicas of frozen-fractured tachyzoites showed intramembranous particles of different densities and sizes on the fractured faces of rhoptry membranes. Both in tachyzoites and bradyzoites, the intramembranous particles were arranged in distinctive parallel arrays that decorated most part of these organelles. Tubulo-vesicular subcompartments and free particles within the rhoptry lumen were observed on freeze-fractured replicas. Cryo-fixed, deep-etched samples showed several pore-like structures localized in the bulb portion. No obvious evidence was found of a possible connection between rhoptries and micronemes.

Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that transfer replicated RNA to assembly sites in Golgi membranes. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER and CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities.

Vacuolar H+ATPase (V-ATPase) accumulates protons inside various intracellular organelles, generating the electrochemical proton gradient required for many vital cellular processes. V-ATPase is a complex enzyme with many subunits that are organized into two domains. The membrane domain that translocates protons contains a proteolipid oligomer of several c subunits and a 100 kDa a subunit. Several a-subunit isoforms have been described that are important for tissue specificity and targeting to different membrane compartments, and could also result in the generation of V-ATPases with different functional properties. In the present report, we have cloned the Torpedo marmorata a1 isoform. This isoform was found to be addressed specifically to nerve endings, whereas VATPases in the neuron cell bodies contain a different a-subunit isoform. In nerve terminals, the V-ATPase membrane domain is present not only in synaptic vesicles but also in the presynaptic plasma membrane, where its density could reach 200 molecules μm–2. This V-ATPase interacts with VAMP-2 and with the SNARE complexes involved in synaptic vesicle docking and exocytosis.

8 pages, 7 figures.-- PMID: 15099572 [PubMed].-- Printed version published Jun, 2004.-- Corrigendum published in: Journal of Structural Biology 148(2): 257 (Nov 2004), http://dx.doi.org/10.1016/j.jsb.2004.06.001, A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum–carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist., This study was supported mainly by funds from DGICYT (Dirección General de Investigación científica y técnica) (Prog. No. 2001-1188-CO2-02).

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.

International audience; The malaria parasite invades the midgut tissue of its mosquito host as a motile form called the ookinete. We have examined the pellicle of the ookinete of Plasmodium gallinaceum by freeze-fracture and quick-freeze, deep-etch electron microscopy. The general organization is analogous to that of invasive stages of other members of Apicomplexa. The pellicle is composed of three membranes: the plasma membrane, and the two linked intermediate and inner membranes, which in the ookinete form one flattened vacuole that is located beneath the plasma membrane. The edges of this vacuole form a longitudinal suture. Beneath the vacuole is found an array of microtubules that are connected to the inner membrane by intramembranous particles. During freeze-fracture, the membranes can split along their hydrophobic planes, thus yielding six fracture faces, each of which displays a characteristic pattern of intramembranous particles. Additionally, we find that the ookinete pellicle differs from all other apicomplexan motile stages by the presence of large pores. These pores are of unknown function, but clearly might constitute a novel pathway for the transport of molecules to and from the cortex, which is independent of the well-described route through the apical micronemal/rhoptry complex. The pores may be the route by which motor proteins or other non micronemal surface proteins are trafficked, such as P25/P28 and SOAP, some of which are implicated in transmission blocking immunity.

In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin alterations during an early stage of Wallerian degeneration. Rat sural nerves were experimentally crushed and animals were sacrificed by transcardiac perfusion 30 h after surgery. Segments of the nerves were processed for routine transmission electron microscopy and freeze-fracture techniques. Our results show that 30 h after the lesion there was asynchrony in the pattern of Wallerian degeneration, with different nerve fibers exhibiting variable degrees of axon disruption. This was observed by both techniques. Careful examination of several replicas revealed early changes in myelin membranes represented by vacuolization and splitting of consecutive lamellae, rearrangement of intramembranous particles and disappearance of paranodal transverse bands associated or not with retraction of paranodal myelin terminal loops from the axolemma. These alterations are compatible with a direct injury to the myelin sheath following nerve crush. The results are discussed in terms of a similar mechanism underlying both axon and myelin breakdown.

A biochemical and structural analysis is presented of fractions that were obtained by a quick and mild solubilization of thylakoid membranes from spinach with the non-ionic detergent n-dodecyl-alpha ,D-maltoside, followed by a partial purification using gel filtration chromatography. The largest fractions consisted of paired, appressed membrane fragments with an average diameter of about 360 nm and contain Photosystem II (PS II) and its associated light-harvesting antenna (LHC II), but virtually no Photosystem I, ATP synthase and cytochrome b(6)f complex. Some of the membranes show a semi-regular ordering of PS II in rows at an average distance of about 26.3 nm, and from a partially disrupted grana membrane fragment we show that the supercomplexes of PS II and LHC II represent the basic structural unit of PS II in the grana membranes. The numbers of free LHC II and PS II core complexes were very high and very low, respectively. The other macromolecular complexes of the thylakoid membrane occurred almost exclusively in dispersed forms. Photosystem I was observed in monomeric or multimeric PS I-200 complexes and there are no indications for free LHC I complexes. An extensive analysis by electron microscopy and image analysis of the CF0F1 ATP synthase complex suggests locations of the delta (on top of the F-1 headpiece) and epsilon subunits (in the central stalk) and reveals that in a substantial part of the complexes the F-1 headpiece is bended considerably from the central stalk. This kinking is very likely not an artefact of the isolation procedure and may represent the complex in its inactive, oxidized form.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

2 H NMR spectroscopy and freeze-fracture electron microscopy were used to compare the transmembrane domains of two Class I protein receptor tyrosine kinases (the EGF receptor and Neu/erbB-2) regarding overall behaviour in fluid lipid bilayer membranes. The 34-residue peptide, EGFRtm, was synthesised to contain the 23 amino acid hydrophobic stretch (Ile622 to Met644) thought to span the membrane of the human EGF receptor, plus the first 10 amino acids (Arg645 to Thr654) of the cytoplasmic domain. Deuterium probes replaced selected 1 H nuclei at sites corresponding to Ala623, Met644, and Val650. The 38-residue peptide, Neutm, was synthesised having the 21 residue hydrophobic stretch (Ile660 to Ile680) calculated to span the membrane in rat Neu/erbB-2, plus residues Lys681 to Thr691 of the contiguous cytoplasmic domain. Deuterium probes replaced selected 1 H nuclei at Ala661, Leu667, and Val676. A third peptide, Neutm*, was also prepared, corresponding to the transmembrane domain of a constitutively-activating Neu/erbB-2 transformant in which Val664 is replaced by Glu: it was deuterated in a manner identical to Neutm. Peptides were studied by 2 H NMR spectroscopy at 1 mol% and 6 mol% in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in POPC containing 33 mol% cholesterol, over the range 12° to 65°C. Overall motion was found to be different for each of the three peptides under a given set of conditions. EGFRtm spectra were characteristic of axially symmetric motion in membranes of POPC alone, and in POPC/cholesterol at 35°C and above. In contrast, spectra of the transmembrane peptides, Neutm and Neutm*, were characteristic of significantly axially asymmetric motion under all conditions studied (and regardless of sample preparation method). Addition of 33% cholesterol to membranes was accompanied by spectral changes consistent with increased formation of peptide dimers/oligomers in all cases. The transformant peptide, Neutm*, showed greater spectral evidence of immobilisation than did the wild type - probably reflecting a greater tendency to form large oligomers. Sequence-related details within the transmembrane domains of Class I receptor tyrosine kinases appear to exert important control over their associations within membranes. Freeze-fracture electron microscopy of the NMR samples demonstrated their liposomal nature. Peptide-related intramembranous particles (IMPs) were present which likely represent oligomers of the transmembrane peptide. IMP size and distribution were similar under a given set of conditions for all three peptides, suggesting that the differences seen by NMR spectroscopy reflect structures smaller than the 2 nm resolution limit of freeze-fracture EM and peptide relationships within its 20 nm accuracy of identifying lateral position.

The epicuticle of infective larvae of Trichinella spiralis represents the interface between this intracellular nematode parasite and the cytosol of mammalian skeletal muscle. The macromolecular structures that make up the epicuticle were studied by freeze-fracture electron microscopy and compositional analysis. Three fracture planes were observed: one with a typical plasma membrane-type bilayer organisation which was overlaid by two extended layers of lipid in an inverted cylindrical configuration. This overall structure remained unchanged in response to variations in temperature between 20 degrees C and 45 degrees C. The lipid cylinders were on average 6.8 nm in diameter, with randomly-associated particles that were not dissociated by high-salt treatment, indicative of hydrophobically associated proteins. The majority of the lipids were non-polar, consisting of cholesterol, cholesterol esters, mono- and tri-glycerides, and free fatty acids. Three major classes of phospholipids were identified: phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. Total lipid extracts did not adopt an inverted cylindrical or micellar configuration on isolation, but formed flat sheets of lamellae as did the purified polar and non-polar fractions of the lipids. Isolated lipids did not undergo thermally-induced polymorphism between 20 degrees C and 60 degrees C and there was no pH dependency of the structures adopted. The fatty acid saturation levels of the phospholipids were compatible with the observation that they did not form polymorphic structures on isolation. We suggest that this unusual configuration is probably stabilised by the associated (glyco)proteins and may be required for selective permeation of nutrients from the host cell cytosol and/or for maintaining the high curvature of the parasite within the cell.

Cryofixation is considered to be the best method for immobilizing biological material in its natural state. In jet-freezing, the specimen typically is sandwiched between two carriers and kept in place while a coolant is moved very rapidly against the opposite surfaces. The JFD 030 jet-freezing device has been used to optimize the operating parameters. The course of the temperature in place of a specimen was measured with thermocouples and recorded by an IBM-compatible personal computer using a specifically developed software program. Mean cooling rates, over the temperature range of 273K to 173K, achievable with different cryogens, including the non-flammable HCFC 124 (SUVA 124-CHClFCF3), were measured under a variety of conditions. The freezing capability of the JFD 030 was evaluated by analyzing transmission electron microscopic results obtained from freeze-substituted plant cells and freeze-fractured cosmetic emulsions. Jet-freezing, despite its limits in freezing thick specimens, can be applied to cell suspensions as well as to semi-thin sections by the use of thin Ti supports, a higher pressure of the nitrogen gas or by a brief pretreatment with a cryoprotectant (e.g., sucrose). In addition, with the non-flammable HCFC 124 in combination with the thin Ti supports, freezing rates similar to those achievable with propane and standard copper supports can be reached giving researchers the chance to use jet-freezing without the dangerous propane.

The water-soluble antineoplastic agent 5-fluoro-2'-deoxyuridine (FUdR) was encapsulated in the water phase of liposomes of different lipid compositions. The retention of this drug upon storage and during contact with plasma was assessed. It was found that, upon refrigeration, diffusion of FUdR across the liposome bilayer was considerably faster when the drug was encapsulated in fluid-type liposomes (egg PC/PS/CHOL) than in solid-type liposomes (DSPC/DPPG/CHOL). With either composition, leakage of the drug from the liposomes was accelerated upon contact with plasma. To achieve improved liposomal retention of the drug, FUdR was converted to a lipophilic prodrug by esterifying the free hydroxyl groups in the deoxyribose moiety with fatty acids of different chain lengths. Thus FUdR-dipalmitate (C-16) and FUdR-dioctanoate (C-8) were synthesized and incorporated in liposomes. The dipalmitoyl derivative could be incorporated upto 13 mol% in solid-type liposomes but to only 2 mol% in fluid-type liposomes. Freeze-fracture electron microscopy revealed no major differences between control liposomes and those containing the prodrug. FUdR-dipalmitate was found to be firmly associated with the liposomal bilayer in both liposome-types: no exchange of the pro-drug with blood constituents or hydrolysis by serum esterases could be registered when the liposomes were incubated with serum. On the other hand, liposome-incorporated FUdR-dioctanoate was found to be readily extracted from the liposomes by serum components (predominantly albumin) and was found to be degraded rapidly by serum esterase activity. The antitumor activity of FUdR-prodrugs was determined using C26 colon adenocarcinoma cells. This cell line was found to be highly sensitive to FUdR. Liposomal FUdR-dioctanoate inhibited cell growth in the same concentration range as unesterified FUdR. FUdR-dipalmitate, however, was more than two orders of magnitude less potent in inhibiting cell proliferation. Its antiproliferative activity was dependent on the liposome-type used: when incorporated in fluid-type liposomes, antiproliferative activity of FUdR-dipalmitate was several-fold higher than in solid-type liposomes. The difference in antitumor activity between FUdR-dipalmitate and FUdR-dioctanoate and between FUdR-dipalmitate in the fluid- and solid-type liposomes could be explained by differences in the rate of hydrolysis of the prodrugs to FUdR by esterase activity in the tumor cells or in the growth medium.

This review of cryofixation by plunge cooling surveys liquid coolants, plunge cooling devices, modeling and applications featuring time resolution. It then focuses on thermocouple experiments and ice crystal analyses. These highlight the effects of the design of specimen holders, the cold gas above the coolant which can freeze the specimen prematurely, specimen size, plunge velocity, coolant depth and ultimate coolant efficiencies. The ice crystal cavity analyses are validated by experiments which monitor the effects of high subzero temperatures on the storage of plunge cooled specimens and an experiment which monitored the rate of cryosubstitution. Ethane was found to be more efficient for plunge cooling metal specimens (bare thermo-couples), exposed hydrated specimens and metal-sandwiched freeze fracture specimens; this suggests that it should be used for plunge freezing, spray freezing and jet freezing.

Screw implants of commercially pure titanium and titanium alloy together with rods of slip-cast dense hydroxyapatite were implanted transfemorally in young adult rats. The femora were harvested after fixation and freeze-fractured in liquid nitrogen to create two tissue fragments of each specimen. The first contained the implant while the second enabled examination of the tissue immediately adjacent to the implant. In all cases, the tissue abutting the implant was a cement line-like layer which separated the implant from the surrounding bone proper. Greater adherence of this interfacial tissue was demonstrated to hydroxyapatite samples compared with the titanium screws, while no adherence was demonstrated to the titanium alloy implants. The differences in adhesion of interfacial tissue to titanium and titanium alloy were attributed, in some areas, to localized increase in surface roughness, and in others to subtle chemical differences between the oxide-covered metal surfaces. The cement line-like layer was composed of globular accretions which were clearly visible on the apparently smooth grain surfaces of the retrieved hydroxyapatite rods. However, modification of the ceramic, during the implantation period, to produce a micropitted surface, also suggested that microtopographical features were implicated in interfacial tissue adhesion.

Studies were undertaken to expand the versatility and the resolution of low temperature conventional and field emission scanning electron microscopy (SEM). The results indicated that simple modified specimen holders, which could be used in conjunction with the commercial cryosystems, allowed one to store specimens for several weeks in liquid nitrogen, either before or after observation in a conventional SEM, without incurring degradation of the surface features. Other modified holders permitted one to move the specimen closer to the final lens or to use the upper secondary electron detector, which is available with some SEMs. Both of these procedures increased the resolution that was attainable with the standard holders. In conventional SEM (CSEM) and field emission SEM (FESEM), holders were also modified to allow one to obtain complementary images of fractured specimens. When a conventional vacuum evaporator equipped with a freeze-etch module was used in conjunction with these holders, specimens could be fractured, etched, shadowed with platinum and coated with carbon before the sample was transferred to the cryostage in the SEM. This procedure increased resolution beyond that obtained with the sputter units in two commercial cryosystems that were used on a CSEM and a FESEM, provided membrane particle resolution in the FESEM and produced a coating or replica that could be recovered and examined in a TEM. These results, which demonstrated how resolution of cryospecimens can be enhanced in CSEM and FESEM, indicated that coating specimens in a high vacuum evaporator provided an alternative procedure that could be used to obtain high resolution images in a FESEM.

The freeze-fracture technique was used to analyse the organization of the plasma membrane, as well as membranes of cytoplasmic organelles, of the pathogenic protozoan Trichomonas vaginalis. Rosettes formed by 4 to 14 intramembranous particles were seen on the fracture faces of the membrane lining the anterior flagella as well as in fracture faces of the plasma membrane enclosing the anterior region of the protozoan and in cytoplasmic organelles. Special organization of the membrane particles were also seen in the region of association of the recurrent flagellum to the cell body.

Cryo-preparation is a reliable technique for the structural investigation of food products in low temperature scanning electron microscopy (SEM). Artifacts, such as, the segregation of water/non-water ingredients, occur during the freezing process by the crystallization of ice; they can be helpful for correct interpretation of visualized details, e.g., the detection of water containing compartments. The size of the segregation structures depends on water concentration and specimen thickness. The condensation of water vapor (ice contamination) is influenced by the specimen temperature and the partial pressure of the water inside the vacuum system. Furthermore, the evaporation (sublimation, etching) of specimen water can be regulated by monitoring the specimen temperature. Sublimation under SEM observation, i.e., "in situ etching" at low acceleration voltage, allows the progress of etching to be observed continuously, prior to the coating of the specimen inside a dedicated cryo-preparation system attached to the SEM. Coating of specimens provides superior structural resolution compared with the observation of uncoated samples. A coating layer of platinum ( ~ 1-2 nm thick), deposited on a cold substrate by planar magnetron sputtering, is almost homogenous and has a density close to that of the solid metal. Its use allows bulk biological specimens to be observed in low temperature SEM with a structural resolution up to the visualization of transmembrane proteins.

With freeze-fracturing sperm cells appear to be fractured preferentially through the plasma membranes. Only few fracture planes through the cytoplasm are found. Both the PF as well as the EF side of the sperm cell plasma membranes show a slightly undulating surface and contain intramembrane particles. The particle distribution is irregular and does not show any clustering. The EF side of the plasmamembrane contains approximately 3 times more particles per μm2 than the PF side.