Isolation and characterization of subcellular membranes of Entamoeba invadens.

A method is described for the isolation of subcellular membranes of Entamoeba invadens. Plasma membranes were obtained by rate centrifugation followed by isopycnic centrifugation on a sucrose gradient. Intact phagolysosomes floated in a 10% sucrose solution providing a simple technique for isolation. Phagolysosomal membranes were collected by isopycnic centrifugation, after lysis of the phagolysosomes. Microsomes were obtained by differential centrifugation. Membrane fractions were examined by electron microscopy, and the contamination of each fraction was determined with marker enzymes. Mg2+-ATPase is associated with the plasma membrane. Acid phosphatase (beta-glycerophosphate) was associated mainly with phagolysosmal membranes. Plasma membranes also contained acid phosphatase activity which hydrolyzes p-nitrophenylphosphate but not beta-glycerophosphate. The localization of the two phosphatases was confirmed cytochemically. Isolated plasma membranes were contaminated with phagolysosomal membranes (15%) and with microsomes (25%). No more than 5% of the phagolysosomal membrane fraction consisted of plasma membranes. Contamination of the microsomes by plasma and phagolysosomal membranes was 10% and 7%, respectively. Plasma membranes and phagolysosomal membranes had a high ratio of cholesterol to phospholipid (0.93 and 1.05 mumol/mumol, respectively). Microsomes were relatively poor in cholesterol (0.39 mumol/mumol). Microsomes, plasma, and phagolysosomal membranes contained increasing amounts of spingolipids (12%, 17%, and 28%). Phagolysosomal membranes had a high percentage of phosphatidylserine but little phosphatidylcholine. Microsomes were rich in phosphatidylcholine (45%). Differences in phospholipid composition between plasma and phagolysosomal membranes are discussed in view of the phagocytic process.

Synthesis and evaluation of rifabutin analogs against Mycobacterium avium and H(37)Rv, MDR and NRP Mycobacterium tuberculosis.

Clinical utility of rifabutin 1 (RBT), a potent antibiotic used in multidrug regimens for tuberculosis (TB) as well as for infections caused by Mycobacterium avium complex (MAC), has been hampered due to dose-limiting toxicity. RBT analogs 2-11 were synthesized and evaluated against M. avium 1581 and Mycobacterium tuberculosis susceptible and resistant strains in vitro. A selection of candidates were also assayed against non-replicating persistent (NRP) M. tuberculosis. Subsequent in vivo studies with the best preclinical candidate drugs 5 and 8, in a model of progressive pulmonary tuberculosis of Balb/C mice infected either with H(37)Rv drug-sensible strain or with multidrug resistant (MDR) clinical isolates, resistant to all primary antibiotics including rifampicin, were performed. The results disclosed here suggest that 5 and 8 have potential for clinical application.

The effect of microfluidization of protein-coated liposomes on protein distribution on the surface of generated small vesicles.

Tetanus toxoid and immunoglobulin G (IgG), model proteins for vaccines and targeting ligands respectively, were covalently coupled to preformed dehydration-rehydration vesicles (DRV) to produce vesicles with surface-bound proteins (DRV-protein) or to preformed small unilamellar vesicles (SUV) which were used to generate DRV with bound protein [(SUV-protein)DRV]. Of the amount of protein employed for coupling (1 mg), 13.8-45.1% was recovered with the liposomes, depending on the type of preparations and the proteins used. Microfluidization of similar DRV-protein or (SUV-protein)DRV for up to 10 cycles led to the formation of smaller vesicles (98-136 mm diameter) which, however, had modestly reduced (estimated as 8.8-21.7%) bound proteins, again depending on the type of preparation and protein used. Treatment of DRV-protein and (SUV-protein) DRV with proteinase revealed that 32.9-45.6% of the total bound protein was exposed on the liposomal surface. With microfluidized liposomes, the proportion of surface-exposed protein increased to 63.1-76.2%. Incubation of intact and microfluidized DRV-IgG and (SUV-IgG) DRV with a protein A-Sepharose 4B CL gel confirmed the presence of IgG on the liposomal surface (47.1-68.4 and 80.5-82.1% of total bound protein respectively). These studies were supplemented with freeze-fracture electron microscopy of (SUV-toxoid)DRV which demonstrated the presence of protein particles (up to 3; 12-14 nm diameter) on the surface of both intact and microfluidized individual liposomes.

Morphological observations on liposomes bearing covalently bound protein: studies with freeze-fracture and cryo electron microscopy and small angle X-ray scattering techniques.

The appearance of protein bound to the surface of intact and microfluidized liposomes and its possible influence on their morphology was examined by freeze-fracture electron microscopy, cryo electron microscopy and small angle X-ray scattering (SAXS) techniques. Results obtained by the two microscopy techniques were in agreement with one another in terms of vesicle size and localization of protein (tetanus toxoid or immunoglobulin G) on the surface of vesicles. Surface-bound protein was observed as particles (10-12 nm diameter) by freeze-fracture electron microscopy and was confirmed by immunogold cryo microscopy. SAXS was shown to be a suitable means to further characterize liposomes with, or without bound protein.

An optimized freeze-fracture replication procedure for human skin.

This in vitro study aimed at substantial modification of the freeze-fracture replication technique (FFRT) which should result in an optimal visualization of the ultrastructure of human skin. The technique was modified in two ways: firstly, the conventional sample holders such as gold cups and copper plates were replaced by silver cylinders (83.5% silver, 16.5% copper) resulting in almost perpendicular cross fractures through the skin. Secondly, the replica cleaning procedure was optimized through the following sequence of treatments. Firstly, a mild tissue destruction was obtained by simultaneous lipid solvation and water extraction with absolute methanol (20 h), followed by protein denaturation with dimethyl sulfoxide (DMSO, 24 h). Subsequently, a final treatment was given using an alkaline sodium hypochlorite solution (20% KOH/13% NaClO; 1:3 v/v, 4 d). After rinsing the replicas for 45 min in aqua bidest, they were mounted on copper grids and examined in the transmission electron microscope (TEM). The combination of the unorthodox fracturing method and the optimized cleaning procedure yielded large, practically undamaged and very clean replicas of near perpendicular cross fractures through human skin. Common handicaps related to current freeze-fracture procedures when applied to skin, such as incomplete cleaning and fragmentation of replicas and oblique or irregular fracturing planes, can largely be avoided in this way. In this paper a complete description of the method is given, and a number of advantages are illustrated with the aid of TEM micrographs.

Freeze-fracture electron microscopy of in vitro reconstructed human epidermis.

Epidermis has been reconstructed in vitro by seeding human keratinocytes on a human dermal substrate in an air-exposed culture. The end product has been examined by freeze-fracture electron microscopy, transmission electron microscopy (TEM) of thin sections, light microscopy, and lipid analysis using thin-layer chromatography. Light microscopic observation of hematoxylin-eosin stained, paraffin embedded cross-sections of the cell culture revealed a strong resemblance to its intact human counterpart, especially with respect to the morphologic organization in basal, spinous, granular, and horny layers. Freeze-fracture electron microscopy and TEM of thin sections generally confirmed the observed resemblances and additionally suggested the presence of lamellar bodies in the stratum granulosum, and of lamellar (lipid) structures between the corneocytes. However, some imperfections were also observed, including some anomalous lipid structures in the intercellular space. Lipid analyses in conjunction with essential fatty acid enrichment studies suggested that the structural anomalies observed in the cultured system may be caused by a lack of linoleyl-ceramides resulting from "immobilization" of linoleyl moieties in the form of triglycerides and phospholipids. In its present form, the air-exposed cell culture already looks very promising as a model for studies of, e.g., skin differentiation disorders such as psoriasis or ichthyosis, studies of the percutaneous penetration and intra(epi)dermal biotransformation of drugs, and skin toxicity screenings. It is furthermore expected that the aforementioned imperfections in the air-exposed cell culture should be avoidable by changing culture conditions such as the relative humidity and the pH, the composition of the medium, or both.

Structure of fully hydrated human stratum corneum: a freeze-fracture electron microscopy study.

The structure of fully hydrated human stratum corneum was investigated by means of freeze-fracture electron microscopy. Mammary and abdominal stratum corneum were incubated for 48 h with phosphate-buffered saline, pH 7.4, occlusively or phosphate buffer, pH 7.4, occlusively and non-occlusively. The micrographs showed the corneocytes aligned parallel to the surface of the stratum corneum embedded in intercellular lipids. The corneocytes were swollen by the uptake of water. New features located in the intercellular lamellar regions were rough structures, water pools, and occasionally vesicle-like structures. The nature of the vesicle-like structures was not completely clear. The presence of water pools, mostly in close contact with the rough structures, suggests that a lipid-water phase separation occurred. The localization of water in the intercellular region and the corneocytes offers new insights into the penetration enhancement property of water (and into the pathways of drug penetration).

Electroperturbation of the human skin barrier in vitro: II. Effects on stratum corneum lipid ordering and ultrastructure.

In transdermal iontophoresis, drugs can be driven across the skin by electrorepulsion, but their transport can also be enhanced by electrical perturbation of the skin barrier. Our objective was to study perturbing effects of electrical current on human stratum corneum lipid fine structure combining techniques including freeze-fracture electron microscopy. Human stratum corneum was subjected to pulsed constant currents, varying from 0.013-13 mA.cm-2. The voltage across the stratum corneum was high-frequency-sampled and s.c. impedence values derived from it. Upon termination of the current, skin samples were rapidly frozen and processed for freeze-fracture electron microscopy or subjected to X-ray diffraction analysis. Initially a rapid decrease of the resistance and, overall, a rapid increase of the capacitances was observed; generally, these effects became more pronounced with increasing current density. Wide- and small-angle X-ray diffractograms of human stratum corneum exposed for 1 h to the highest current indicated a disordering of both the lateral packaging arrangement and long-range lamellar stacking of the intercellular lipids of stratum corneum. Furthermore, an increase in the stratum corneum hydration level as a result of electrical current application was observed. On electron micrographs of freeze-fracture replicas of human stratum corneum, exposed for 1 h to current densities between 0.013 and 13 mA.cm-2, perturbations of the intercellular lipid structure were observed in accordance with the results of X-ray diffraction; these perturbations aggravated with increasing current density. Together, the data suggest that both the lateral and the longitudinal disordering of the intercellular lipids observed with X-ray diffraction may be responsible for the appearance of perturbed structures observed with freeze-fracture electron microscopy. The lipid disordering may be due to polarization of the lipid head groups induced by the electrical field, followed by mutual repulsion.

Teaching law students in the medical schools.

Traditional efforts in the law schools to improve interprofessional communication between the medical and legal professions have been confined to abstract case studies and other academic materials, sometimes augmented with visits and demonstrations by physicians in the classroom. This paper describes one approach to broaden the prespective of law students during their training by permitting them to experience directly the atmosphere of the teaching hospital and the sociology of surgery practice in that environment. The experiment has proved beneficial to the young members of both professions.

The effect of methylated beta-cyclodextrins on the tight junctions of the rat nasal respiratory epithelium: electron microscopic and confocal laser scanning microscopic visualization studies.

The nasal absorption enhancer randomly methylated beta-cyclodextrin (RAMEB) is thought to increase the paracellular permeability of the nasal epithelium by opening of the tight junctions. The effects of RAMEB on the cytoskeleton of the rat nasal epithelium in vivo were determined by confocal laser scanning microscopy (CLSM). The effects on the tight junctions of the rat nasal epithelium were also investigated, using transmission electron microscopy (TEM) of thin sections. The effects of RAMEB were compared with those of the absorption enhancer sodium taurodihydrofusidate (STDHF). Fifteen minutes after nasal administration of 2% RAMEB in vivo, the distribution of cytoskeletal actin was comparable to the untreated control, suggesting that RAMEB does not cause opening of the tight junctions via cytoskeletal interactions. In contrast, administration of 1% STDHF resulted in changes in the actin staining. Furthermore, with TEM severe damage of the nasal epithelium was observed after treatment with 1% STDHF. Ultrastructural changes of the tight junctions were not apparent in TEM sections after treatment with 2% RAMEB. In conclusion, CLSM and TEM are suitable methods to visualize the effects of absorption enhancers on nasal epithelial morphology.

Osmotic behavior of erythrocytes from patients with a major affective disorder. A freeze-fracture electron microscopic study.

Previously we have demonstrated a state-dependent decrease in the number of membrane vesicles in erythrocytes from patients with a major depressive episode. We now report an increase in the number of membrane vesicles during a manic episode as well as a reduction during lithium treatment and we also present data suggesting that the number of erythrocyte membrane vesicles in the affective disorders is dependent on osmotic shrinkage due to the freezing for freeze-etch electron microscopy. Although caution is required since the interrater reliability of the measurement of osmotic strain was insufficient in the mid range where it was tested, we do not think this invalidates the differences in osmotic strain found in the low and high ranges during depressive and manic episodes respectively. These findings warrant the use of more precise techniques in studies of the osmotic behavior of erythrocytes from patients with a major affective disorder.

Decreased erythrocyte membrane elevations in patients with a major depressive episode.

Washing of erythrocytes from healthy volunteers in an isotonic sodium chloride solution at pH 5.6 results in the occurrence of plasma membrane elevations as observed in freeze-etch electron microscopy. This is prevented by anion permeability inhibiting agents. In the absence of these agents a reduced number of elevations was found in 23 of 36 patients with a major depressive episode. This reduced capacity to form membrane elevations was positively correlated with a genetic vulnerability, defined as an admission to a psychiatric hospital in first-degree relatives.

Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin.

Tape stripping of human stratum corneum is widely used as a method for studying the kinetics and penetration depth of drugs. Several factors can influence the quantity of stratum corneum that is removed by a piece of tape, such as the manner of tape stripping, the hydration of the skin, cohesion between cells, body site and interindividual differences. However, few data are available about the influence of furrows in the human epidermis on the tape-stripping technique. In this study, we investigated the efficacy of tape stripping in removing complete cell layers from the superficial part of the human stratum corneum. A histological section of skin that was tape-stripped 20 times clearly showed nonstripped skin in the furrows, indicating persistent incomplete tape stripping. Replicas of tape-stripped skin surface demonstrated that even after removing 40 tape strips the furrows were still present. We validated the tape-stripping method further with X-ray microanalysis in the mapping mode by scanning electron microscopy, using a TiO2-containing compound as a marker. TiO2 applied to the skin before the tape-stripping procedures was still present after the tenth tape strip, and was specifically located on the rims of the furrows. We emphasize that results from studies using the tape-stripping method have to be viewed from the perspective that cells on one tape strip of the stratum corneum may be derived from different layers, depending on the position of the tape strip in relation to the slope of the furrow, and such results should be interpreted with considerable caution.

Efficacy of micronized titanium dioxide-containing compounds in protection against UVB-induced immunosuppression in humans in vivo.

Micronized pigment-containing sunscreens may provide a good alternative to chemical sunscreens in protection against ultraviolet (UV) B-induced immunosuppression. The metal particles in these products are likely to remain on the skin surface where they can offer broadband protection for both the UVA and UVB regions. We have tested the protective capacity of three titanium dioxide (TiO2)-containing compounds in humans in vivo. The effect on sunburn cell formation has been investigated using transmission electron microscopy, while the mixed epidermal cell lymphocyte reaction (MECLR) has been used as a model for immunosuppression. Furthermore, the influence of titanium on the integrity of the stratum corneum barrier (intercellular lipids and desmosomes) has been examined using freeze fracture electron microscopy. We find that all three compounds protect against sunburn cell formation. The immunoprotection studies show that one of the three compounds does not prevent UVB-induced changes of the MECLR responses. Application of this compound without subsequent UVB irradiation also induces a significant decrease of the MECLR responses. Moreover, the same compound affects the intercellular lipid layers, and desmosomes cannot be detected. The deleterious effect of this compound is probably caused by an incomplete hydrolysis during the TiO2 synthesis. Our findings indicate that micronized pigment-containing compounds can offer good protection against short-term UVB-induced immunomodulation in humans in vivo. However, accurate screening of the synthesis of these compounds is a prerequisite for their safe use as sunscreening agents in human subjects.

Physicochemical properties of endotoxins in large volume parenterals.

Endotoxins of Escherichia coli, Salmonella abortus equi and Serratia marcescens were examined using freeze-etch electronmicroscopy and light scattering techniques, with emphasis on their aggregation state. These endotoxins show distinct features of particle size and mass and they may be affected by the presence or absence of Ca2+ ions. No important differences were observed in the particle characteristics of the respective endotoxins in various infusion fluids.

Visualization of enhancing effects of bile salts on buccal penetration.

The enhancing effects of bile salts on buccal penetration was investigated in vitro using porcine buccal mucosa, correlating permeability changes with histological effects. The permeability of the buccal mucosa to the model compound fluorescein isothiocyanate (FITC) was studied in the presence and absence of bile salts. Light microscopy, freeze-fracture electron microscopy and confocal laser scanning microscopy were used in order to investigate the interaction between the bile salts and the buccal epithelium. A significant increase in permeation of FITC was obtained after co-administration with bile salts. After 4h treatment, bile salts (at a concentration of 0.1M) caused a loss of distal layers in the epithelium and a split of the epithelium from the connective tissue. The results of freeze-fracture studies show that the bile salts affect the cytoplasmic domain of the buccal epithelium. Due to the bile salt treatment, the mode of fracture was altered in such a way that cell membranes were almost absent. However, no differences were observed between the enhancing effects of dihydroxy and trihydroxy bile salts, either with the transport rate or with the histological studies.

An improved specimen table for the Balzers freeze etching system BAF 400.

A time saving, simple and inexpensive adaptation of a Balzers specimen table is described. This specimen table is used in combination with the small specimen holders originally developed for the Denton Freeze-etching module. They are in particular suited for cell suspensions, and are easy to handle for freezing procedures. Ten replicas are produced routinely per freeze fracture run, with the aid of this combined system.

Lipid composition and barrier function of human skin after grafting onto athymic nude mice.

The barrier properties of human epidermis grafted for 1-3 months onto nude mice are compared with normal human skin. Beside penetration studies with tritiated water and measurements of transepidermal water loss (TEWL), we analyzed the epidermal lipids by high-performance thin layer chromatography and evaluated the ultrastructure of the intercorneocyte lipid arrangement by freeze fracture electron microscopy (FFEM). The permeability of human skin for tritiated water and the TEWL exhibit no significant changes after grafting onto nude mice. FFEM analysis showed that grafted epidermis has the same morphological pattern as normal human epidermis. Regular desmosomes and lamellar lipid structures are present. Grafting did not qualitatively affect the lipid composition of human epidermis. Ceramides which contribute largely to the barrier function, have the same distribution profile.