Visualization studies of human skin in vitro/in vivo under the influence of an electrical field.

The aim of this study was to investigate the local changes in the ultrastructure of human skin after iontophoresis, using cryo-scanning, transmission and freeze fracture electron microscopy in human skin in vitro and in vivo. Human dermatomed skin was subjected to passive diffusion for 6 hours followed by nine hours of iontophoresis at 0.5 mA/cm2. The skin was processed and examined using both cryo-scanning electron microscopy (Cryo-SEM) and transmission electron microscopy (TEM). In addition, iontophoresis patches were applied to healthy volunteers for 3.5h with 0.5h of passive delivery followed by 3h of iontophoresis at a current density of 0.25 mA/cm2. Subsequently, a series of tape stripping were performed, which were visualized by freeze fracture transmission electron microscopy (FFTEM). In vitro, the cryo-scanning electron microscopy study revealed that electric current induced changes in the water distribution in the stratum corneum. Transmission electron microscopy showed no local changes in the ultrastructure of the stratum corneum; however, layers of detached corneocytes were frequently observed especially at the anodal site. In vivo, there was no evidence of perturbation of the stratum corneum lipid organization; however, changes in the fracture were noticed deeper in the stratum corneum at the anodal side, indicating a weakening of the desmosomal structure. The in vitro/in vivo studies suggest that iontophoresis results in the formation of intercellular water pools (in vitro observation) and a weakening of the desmosomal structure (in vivo observation) only in the upper part of the stratum corneum. However, no changes in the lipid organization were observed in vitro and in vivo at the current densities of 0.5 and 0.25 mA/cm2, respectively. Therefore, even at relatively high current densities, no drastic changes in the ultrastructure of the stratum corneum are observed. As far as structural changes in stratum corneum are concerned iontophoresis is therefore a safe method at the experimental conditions we used.

Field-emission scanning electron microscopy analysis of morphology and enzyme distribution within an industrial biocatalytic particle.

Field-emission scanning electron microscopy (FESEM) was used in a technical feasibility study to obtain insight into the internal morphology and the intraparticle enzyme distribution of Assemblase, an industrial biocatalytic particle containing immobilized penicillin-G acylase. The results were compared with previous studies based on light and transmission electron microscopic techniques. The integrated FESEM approach yielded the same quantitative results as the microscopic techniques used previously. Given this technical equivalence, the integrated approach offers several advantages. First, the single preparation method and detection system avoids interpretation discrepancies between corresponding areas that were examined for different properties with different detection techniques in different samples. Second, the specimen size suitable for whole particle study is virtually unlimited, which simplifies sectioning and puts less stringent demands on the embedding technique. Furthermore, the sensitivity toward enzyme presence and distribution increases because the epitopes inside thick sections become available for labeling. Quick and unambiguous analysis of the relation between particle morphology and enzyme distribution is important because this information may be used in the future for the design of enzyme distributions in which the particle morphology can be used as a control parameter.

Combined chemical and electrical enhancement modulates stratum corneum structure.

In a previous in vitro study it has been shown that pretreatment with a water-based surfactant formulation results in a two-fold increase in transdermal iontophoretic transport of R-apomorphine compared to iontophoresis only. The aim of the study presented in this paper was to unravel the mechanisms involved in the increased iontophoretic delivery. Freeze fracture electron microscopy and cryo-scanning electron microscopy were used to visualise the ultrastucture of human stratum corneum after (i) application of the surfactant formulation, (ii) iontophoresis and (iii) application of the surfactant formulation followed by iontophoresis. Non-occlusive application of the surfactant formulation did not exert any detectable changes in the ultrastructure of the stratum corneum, except for swelling of the outermost corneocyte layers. Application of a current density of 0.5 mA/cm(2) for 9 h induced a swelling of the corneocytes and the formation of water pools that were occasionally present in the intercellular regions. Application of the surfactant formulation followed by iontophoresis resulted in a further swelling of the corneocytes and a frequent presence of water pools in the intercellular regions throughout the whole stratum corneum. The observed changes in the ultrastructure of the stratum corneum can explain the increased R-apomorphine transport during iontophoresis.

An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores.

AIMS: To identify and characterize an antifungal compound produced by Bacillus subtilis YM 10-20 which prevents spore germination of Penicillium roqueforti. METHODS AND RESULTS: The antifungal compound was isolated by acid precipitation with HCl. This compound inhibited fungal germination and growth. Identification by HPLC and mass spectrometry analysis showed high similarity to iturin A. Permeabilization and morphological changes in P. roqueforti conidia in the presence of the inhibitor were revealed by fluorescence staining and SEM, respectively. CONCLUSOINS: The iturin-like compound produced by B. subtilis YM 10-20 permeabilizes fungal spores and blocks germination. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescence staining in combination with flow cytometry and scanning electron microscopy are efficient tools for assessing the action of antifungal compounds against spores. Iturin-like compounds may permeabilize fungal spores and inhibit their germination.

Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour.

Storage of neem (Azadirachta indica) seeds is difficult because of their sensitivity to chilling stress at moisture contents (MC) > or =10% or imbibitional stress below 10% MC. The hypothesis was tested that an elevated gel-to-liquid crystalline phase transition temperature (Tm) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ Tm of hydrated membranes was between 10 degrees C and 15 degrees C, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 degrees C, but remained high at 25 degrees C. The loss of viability coincided with an increased leakage of K+ from the embryos upon imbibition and with an increased proportion of cells with injured plasma membranes. Freeze-fracture replicas of plasma membranes from chilled, hydrated axes showed lateral phase separation and signs of the inverted hexagonal phase. Dehydrated embryos were sensitive to soaking in water, particularly at low temperatures, but fresh embryos were not. After soaking dry embryos at 5 degrees C (4 h) plus 1 d of further incubation at 25 degrees C, the axis cells were structurally disorganized and did not become turgid. In contrast, cells had a healthy appearance and were turgid after soaking at 35 degrees C. Imbibitional stress was associated with the loss of plasma membrane integrity in a limited number of cells, which expanded during further incubation of the embryos at 25 degrees C. It is suggested that the injuries brought about by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic Tm (10-15 degrees C), are caused by gel phase formation.

The competence to acquire cellular desiccation tolerance is independent of seed morphological development.

Acquisition of desiccation tolerance and the related changes at the cellular level in wheat (Triticum aestivum cv. Priokskaya) kernels during normal development and premature drying on the ear were studied using a spin probe technique and low temperature scanning electron microscopy. During normal development, the ability of embryos to germinate after rapid drying and rehydration was acquired after completion of morphological development, which is a few days before mass maturity. The acquisition of desiccation tolerance, as assessed by germination, was associated with an upsurge in cytoplasmic viscosity, the onset of accumulation of protein and oil bodies, and the retention of membrane integrity upon dehydration/rehydration. These features were also used to assess cellular desiccation tolerance in the cases when germination could not occur. Slow premature drying was used to decouple the acquisition of cellular desiccation tolerance from morphogenesis. Upon premature drying of kernels on the ears of plants cut at 5 d after anthesis, desiccation-tolerant dwarf embryos were formed that were able to germinate. When plants were cut at earlier stages poorly developed embryos were formed that were unable to germinate, but cellular desiccation tolerance was nevertheless acquired. In such prematurely dried kernels, peripheral meristematic endosperm cells had already passed through similar physiological and ultrastructural changes associated with the acquisition of cellular desiccation tolerance. It is concluded that despite the apparent strong integration in seed development, desiccation tolerance can be acquired by the meristematic cells in the developing embryo and cambial layer of endosperm, independently of morphological development.

Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine Peyer's patches.

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine in the gut and low uptake in the lymphoid tissue of the gastrointestinal tract still complicate the development of oral vaccines. In this study chitosan microparticles were prepared and characterized with respect to size, zeta potential, morphology and ovalbumin-loading and -release. Furthermore, the in vivo uptake of chitosan microparticles by murine Peyer's patches was studied using confocal laser scanning microscopy (CLSM). Chitosan microparticles were made according to a precipitation/coacervation method, which was found to be reproducible for different batches of chitosan. The chitosan microparticles were 4.3+/-0.7 microm in size and positively charged (20+/-1 mV). Since only microparticles smaller than 10 microm can be taken up by M-cells of Peyer's patches, these microparticles are suitable to serve as vaccination systems. CLSM visualization studies showed that the model antigen ovalbumin was entrapped within the chitosan microparticles and not only associated to their outer surface. These results were verified using field emission scanning electron microscopy, which demonstrated the porous structure of the chitosan microparticles, thus facilitating the entrapment of ovalbumin in the microparticles. Loading studies of the chitosan microparticles with the model compound ovalbumin resulted in loading capacities of about 40%. Subsequent release studies showed only a very low release of ovalbumin within 4 h and most of the ovalbumin (about 90%) remained entrapped in the microparticles. Because the prepared chitosan microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. Initial in vivo studies demonstrated that fluorescently labeled chitosan microparticles can be taken up by the epithelium of the murine Peyer's patches. Since uptake by Peyer's patches is an essential step in oral vaccination, these results show that the presently developed porous chitosan microparticles are a very promising vaccine delivery system.

Field-emission scanning electron microscopy of the internal cellular organization of fungi.

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.

Automated electron tomography of the septal pore cap in Rhizoctonia solani.

Dolipore septa and septal pore caps (SPCs) in filamentous basidiomycetes may play an important role in maintaining the integrity of hyphal cells. We have investigated the ultrastructure of the dolipore septum and the SPC in Rhizoctonia solani hyphal cells after high-pressure freezing, freeze substitution, and Spurr embedding. We visualized the SPC with associated cell ultrastructures in three dimensions by automated electron tomography of thick-sectioned cells, followed by 3D tomographic reconstructions. Using these methods we were able to document the passage of mitochondria through the SPC, small tubular membranous structures at the entrance of the septal pore channel, filamentous structures connecting the inner side of the SPC with pore-plugging material, thin filaments anchoring the pore-plugging material with the plasma membrane, small vesicles attached to the plugging material, and tubular endoplasmic reticulum continuous with the base of the SPC. We hypothesize that the SPC, the filamentous structures, the plugging material, and the endoplasmic reticulum act in a coordinated fashion to maintain cellular integrity, intercellular communication, and the transport of solutes and cell organelles in the filamentous fungus R. solani.

The second step of the biphasic endosperm cap weakening that mediates tomato (Lycopersicon esculentum) seed germination is under control of ABA.

The role of abscisic acid (ABA) in the weakening of the endosperm cap prior to radicle protrusion in tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds was studied. The endosperm cap weakened substantially in both water and ABA during the first 38 h of imbibition. After 38 h the force required for endosperm cap puncturing was arrested at 0.35 N in ABA, whereas in water a further decrease occurred until the radicle protruded. During the first 2 d of imbibition endo-beta-mannanase activity was correlated with the decrease in required puncture force and with the appearance of ice-crystal-induced porosity in the cell walls as observed by scanning electron microscopy. Prolonged incubation in ABA resulted in the loss of endo-beta-mannanase activity and the loss of ice-crystal-induced porosity, but not in a reversion of the required puncture force. ABA also had a distinct but minor effect on the growth potential of the embryo. However, endosperm cap resistance played the limiting role in the completion of germination. It was concluded that (a) endosperm cap weakening is a biphasic process and (b) inhibition of germination by ABA is through the second step in the endosperm cap weakening process.

Cryo-planing for cryo-scanning electron microscopy.

In the past decade, investigators of cryo-planing for low-temperature scanning electron microscopy (cryo-SEM) have developed techniques that enable observations of flat sample surfaces. This study reviews these sample preparation techniques, compares and contrasts their results, and introduces modifications that improve results from cryo-planing. A prerequisite for all successful cryo-planing required a stable attachment of the specimen to a holder. In most cases, clamping with a screw mechanism and using indium as space-filler sufficed. Once this problem was solved, any of three existing cryo-planing methods could be used to provide successful results: cryo-milling, microtomy in a cold room, and cryo-ultramicrotomy. This study introduces modifications to the cryo-planing technique that produces flat surfaces of any desired plane through a specimen. These flat surfaces of frozen, fully hydrated samples can be used to improve observations from cryo-SEM as well as to enhance results from x-ray microanalysis and (digital) image analysis. Cryo-planing results of chrysanthemum (Dendranthema x grandiflorum Tzvelev) stems, hazel (Corylus avelane L.) stems, and repeseed (Brassica napus L.) pistils are presented to illustrate the use of the planing method on fibrous, hard, and delicate materials, respectively.

Characterization of Membrane Properties in Desiccation-Tolerant and -Intolerant Carrot Somatic Embryos.

In previous studies, we have shown that carrot (Daucus carota L.) somatic embryos acquire complete desiccation tolerance when they are treated with abscisic acid during culture and subsequently dried slowly. With this manipulable system at hand, we have assessed damage associated with desiccation intolerance. Fast drying caused loss of viability, and all K+ and carbohydrates leached from the somatic embryos within 5 min of imbibition. The phospholipid content decreased by about 20%, and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation was unaltered, irrespective of the drying rate. These results indicate that, during rapid drying, irreversible changes occur in the membranes that are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fracture study and by Fourier transform infrared spectroscopy. Rapidly dried somatic embryos had clusters of intramembraneous particles in their plasma membranes, and the transition temperature of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended [beta]-sheet conformation and had a reduced proportion of [alpha]-helical structures. In contrast, the slowly dried somatic embryos had irregularly distributed, but non-clustered, intramembraneous particles, the transition temperature was below room temperature, and the membrane proteins were not aggregated in a [beta]-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot somatic embryos is indirectly caused by an irreversible phase separation in the membranes due to de-esterification of phospholipids and accumulation of free fatty acids.

Biological sulfate reduction using synthesis gas as energy and carbon source.

Biological sulfate reduction was studied in laboratory-scale gas-lift reactors. Synthesis gas (gas mixtures of H(2)/CO/CO(2)) was used as energy and carbon source. The required biomass retention was obtained by aggregation and immobilization on pumice particles. Special attention was paid to the effect of CO addition on the sulfate conversion rate, aggregation, and aggregate composition.Addition of 5% CO negatively affected the overall sulfate conversion rate; i.e., it dropped from 12-14 to 6-8 g SO(2-) (4)/L day. However, a further increase of CO to 10 and 20% did not further deteriorate the process. With external biomass recycling the sulfate conversion rate could be improved to 10 g SO(2-) (4)/L day. Therefore biomass retention clearly could be regarded as the rate-limiting step. Furthermore, CO affected the aggregate shape and diameter. Scanning electron microscopy (SEM) photographs showed that rough aggregates pregrown on H(2)/CO(2) changed into smooth aggregates upon addition of CO. Addition of CO also changed the aggregate Sauter mean diameter (d(32)) from 1.7 mm at 5% CO to 2.1 mm at 20% CO. After addition of CO, a layered biomass structure developed. Acetobacterium sp. were mainly located at the outside of the aggregates, whereas Desulfovibrio sp. were located inside the aggregates.

Ultrastructural changes of Arabidopsis thaliana pollen during final maturation and rehydration.

Several ultrastructural changes occur during dehydration and subsequent rehydration of Arabidopsis thaliana pollen. The cytoplasmic channels, present in the outer part of the intine of the mature, dehydrating pollen grain, degenerate and develop into electron-dense inclusions. At the same time a large quantity of electron-dense material is deposited in the cavities of the exine. A large number of vesicles is produced in the vegetative cell, and they become predominantly located in the peripheral region near the intine. Starch of amyloplasts is consumed and the lipid bodies which originally surround the sperm cells become randomly distributed. In addition, the individual lipid bodies become enveloped by single rough endoplasmic reticulum cisterns.

Effects of cigarette smoke condensate and 12-O-tetradecanoylphorbol-13-acetate on gap junction structure and function in cultured cells.

The effects of cigarette smoke condensate (CSC) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on gap junction structure, quantity and function were investigated. Gap junction morphology was studied in rotary-shadowed freeze-fracture replicas of primary chick embryo hepatocytes. CSC (24 micrograms/ml) induced a strong decrease of gap junction areas; within 6 h the areas were reduced by greater than 60%. In the first 3 h of exposure, TPA (100 ng/ml) also reduced gap junction areas, but in the next 3 h a partial recovery was observed. Protoplasmic fracture face centre-to-centre particle spacings were used as a measure for gap junction coupling. CSC had a slow (although not significant) reducing effect on particle spacings, while TPA induced a reduction from 10.6 nm (control) to 10.0 nm within 3 h, indicating a reduction of coupling. Gap junctions were quantified in thin sections of cultured chick embryo hepatocytes, V79 fibroblasts, and co-cultivated hepatocytes and V79 cells. CSC did not influence gap junction numbers in any of these cultures, while TPA treatment caused a disappearance of gap junctions between hepatocytes and between hepatocytes and V79 cells in the first 12 h of cultivation. In the following 36 h a slow recovery could be observed. Gap junctions between V79 cells had already disappeared within 30 min. Metabolic co-operation between hepatocytes and hypoxanthine-guanine phosphoribosyltransferase-deficient V79 cells was quickly and continuously blocked by CSC over 27 h, whereas the phorbol ester induced a transient block. The dissimilar effects of these compounds on both gap junction structure and function indicate that they act via different mechanisms. The finding that CSC did not inhibit phorbol ester protein kinase C binding and did not activate this protein kinase in vitro supports this hypothesis.