Freeze-fracturing Dotaz Zobrazit nápovědu
In this study we present an innovative method for the preparation of fully hydrated samples of microbial biofilms of cultures Staphylococcus epidermidis, Candida parapsilosis and Candida albicans. Cryo-scanning electron microscopy (cryo-SEM) and high-pressure freezing (HPF) rank among cutting edge techniques in the electron microscopy of hydrated samples such as biofilms. However, the combination of these techniques is not always easily applicable. Therefore, we present a method of combining high-pressure freezing using EM PACT2 (Leica Microsystems), which fixes hydrated samples on small sapphire discs, with a high resolution SEM equipped with the widely used cryo-preparation system ALTO 2500 (Gatan). Using a holder developed in house, a freeze-fracturing technique was applied to image and investigate microbial cultures cultivated on the sapphire discs. In our experiments, we focused on the ultrastructure of the extracellular matrix produced during cultivation and the relationships among microbial cells in the biofilm. The main goal of our investigations was the detailed visualization of areas of the biofilm where the microbial cells adhere to the substrate/surface. We show the feasibility of this technique, which is clearly demonstrated in experiments with various freeze-etching times.
- Klíčová slova
- Biofilm, Candida, Freeze-fracturing, High-pressure freezing (HPF), Staphylococcus, cryo-SEM,
- MeSH
- biofilmy MeSH
- Candida albicans ultrastruktura MeSH
- Candida parapsilosis ultrastruktura MeSH
- elektronová kryomikroskopie metody MeSH
- extracelulární matrix ultrastruktura MeSH
- mikroskopie elektronová rastrovací metody MeSH
- mrazové lámání metody MeSH
- Staphylococcus epidermidis ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The objective of the present paper was to describe the topographic orientation of intramembraneous particles (IMP) in the membranes of freshly ejaculated boar spermatozoa applying the method of freeze fracturing. Disc clusters of IMP's could be distinguished in the acrosome-covering plasma membrane (PF). The border of the head to a distance of about 0.3 micron seemed to contain no IMP's (Figs. 1, 2). In the postacrosomal region in an anterior direction from the posterior ring the IMP's were found to be arranged in palissade slant rows. Statistical measurements of 20 spermatozoa (Tab. I) indicated that the slant rows extended to the greatest distance in the lateral part of the head, up to 0.67 micron (+/- 1.45-0.39) from the posterior ring. In the middle of the head the rows extended to a distance of 0.18 micron (+/- 0.10-0.42). The transition spot between the zone of sparse IMP's and the zone of densely arranged IMP's was at a distance of 1.63 microns (+/- 1.87-0.69) in an anterior direction from the posterior ring (Figs. 3, 4). In the flagellum in the plasma membrane (PF) the first spot of an ample occurrence of IMP's is located in the first mitochondrion in the spiral. The IMP clusters follow the cicumferential orientation of mitochondria in the mitochondrial spiral. The IMP's were missing in the spaces between the spiral coils (Fig. 5). The membranes of the mitochondria contain a large amount of IMP's. This activity is transferred to the clinging part of the plasma membrane (Fig. 6). A larger accumulation of IMP's can be seen at the spot where the plasma membrane covers the annulus (Fig. 7). In the plasma membrane of the main segment of the flagellum the IMP's are distributed irregularly in PF, and also in EF (Fig. 8). The EF surfaces of the plasma membrane had in general an indistinct structural organisation of IMP's. The distribution of IMP's in acrosomal membranes was found to be irregular. The postacrosomal lamina in the freeze fracturing did not look like a morphologically identifiable structure. In the nuclear envelope delimiting the posterior nuclear space, nuclear pores could be identified (Fig. 9). No nuclear pores could be seen in the nuclear envelope clinging to the posterior part of the nucleus. At this spot smaller IMP's were observed which could be a part of the proteins of the filaments connecting the concave surface of the head with the convex articular surface of the flagellum. These filaments connect the head and the flagellum (Fig. 10).(ABSTRACT TRUNCATED AT 400 WORDS)
- MeSH
- buněčná membrána ultrastruktura MeSH
- mrazové lámání * MeSH
- prasata anatomie a histologie MeSH
- spermie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
Freeze-fracture electron microscopy of a pathogenic dimorphic fungus Sporothrix schenckii revealed planar views of cell structures corresponding to those described already on thin sections. In addition to the characteristic differences in cell wall thickness between conidia, yeast forms and filaments, variations in plasma membrane invaginations were found. In conidia the invaginations were short and abundant, while in yeast forms they were scarce and longer. The plasma membrane of the filaments was smooth without invaginations. No differences were found in the frequency of intramembrane particles among the three forms. In the region of the septal pore the particles were circularly arranged with a characteristic partitioning on the P and E fracture faces.
Freeze-fracture replicas of the plasma membrane and tight junctions (Tj) of intestinal epithelial cells were studied in Tilapia nilotica fish exposed to the pyrethroid insecticide, neopybuthrin. Exposing fishes to different repeated concentrations of 1/2 LC50 of neopybuthrin caused a significant decrease in the population density of IMPs in P- and E-faces. Tight junctions were also affected by neopybuthrin treatment. They appeared fragmented and discontinued, and their strands were fewer in number as compared with controls. Since the structure and number of Tj are major determinants of epithelial permeability, it is postulated that neopybuthrin treatment may affect the intestinal permeability of T. nilotica.
- MeSH
- epitel účinky léků ultrastruktura MeSH
- insekticidy farmakologie MeSH
- mrazové lámání MeSH
- pyrethriny farmakologie MeSH
- střevní sliznice účinky léků ultrastruktura MeSH
- Tilapia * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- insekticidy MeSH
- neopybuthrin MeSH Prohlížeč
- pyrethriny MeSH
- MeSH
- dřeň nadledvin ultrastruktura MeSH
- elektronová mikroskopie MeSH
- inbrední kmeny potkanů MeSH
- krysa rodu Rattus MeSH
- mrazové lámání MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Under the light microscope the chitin-protein layer of egg-shells in ascarids appears to be a regular, hyaline and nonstructural layer of 1.5 to 2.00 microns in thickness. The outer uterine layer is usually removed during the preparation. The lipid (ascaroside) layer covers the inner surface of the chitinous layer and seems to be irregularly undulated and regularly thick over the whole surface, with the thickness up to 1 micron. In electron micrographs the fibrous structure of the lipid layer is not evident as a rule. This is probably due to washing the lipids away from this layer during the dehydration of deeper layers of egg-shells that are imperfectly fixed with glutaraldehyde. A very low permeability of the egg-shells is typical of geohelminth eggs. The layer lipid shows a distinct lamellate structure only after a prolonged fixation with osmium at higher temperature. This is supported by the studies using the method of freeze-fracturing.
- MeSH
- Ascaris ultrastruktura MeSH
- chitin MeSH
- elektronová mikroskopie MeSH
- lipidy MeSH
- mrazové lámání MeSH
- ovum ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chitin MeSH
- lipidy MeSH
The purpose of this work is to investigate the entrapment of protein molecules in cubosomic nanocarriers that are sterically stabilized by an amphiphilic poly(ethylene glycol) (PEG) derivative. Toward that aim, the mechanism of fragmentation of a self-assembled, PEGylated cubic lipid phase into nanoparticles (NPs) is investigated in excess aqueous medium. The molar ratio between the cubic-phase-forming lipid monoolein (MO) and its PEGylated derivative (MO-PEG(2000)) is selected as to favor the formation of inverted-type liquid-crystalline (LC) structures (permitting one to reveal the stages of the fragmentation and bicontinuous membrane NP assembly process) rather than a phase transformation to lamellar or micellar phases. The PEGylated amphiphile considerably affects the interfacial curvature of the cubic lipid membrane and, under agitation, contributes to the fragmentation of the bicontinuous cubic lattice into NPs. Freeze-fracture electron microscopy (FF-EM), quasi-elastic light scattering (QELS), and confocal laser scanning fluorescence microscopy (CLSFM) are applied for determination of the NPs' sizes, inner organization, and stability with regard to a thermal stimulus. Entrapped protein molecules can essentially stabilize the cubosomic particles (proteocubosomes), which display well-defined inner organization of nanochannels in their freeze-fracture planes. The protein α-chymotrypsinogen A is studied in proteocubosome dispersions by means of far-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. It is suggested that the protein molecules are entrapped in the interior of the PEGylated cubosomes via a "nanopockets" mechanism. The LC PEGylated proteocubosomes offer new possibilities for investigation of protein loading in sterically stabilized ("Stealth") nanostructured lipid carriers, which differ from Poloxamer-stabilized isasomes.
- MeSH
- chymotrypsinogen chemie MeSH
- cirkulární dichroismus metody MeSH
- elektronová mikroskopie metody MeSH
- glyceridy chemie MeSH
- kapalné krystaly chemie MeSH
- konfokální mikroskopie MeSH
- mrazové lámání metody MeSH
- nanočástice chemie ultrastruktura MeSH
- poloxamer chemie MeSH
- polyethylenglykoly chemie MeSH
- synchrotrony MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chymotrypsinogen MeSH
- glyceridy MeSH
- monoolein MeSH Prohlížeč
- poloxamer MeSH
- polyethylenglykoly MeSH
- voda MeSH
The main types of ultrastructures found in the freeze-fracture faces of Acholeplasma laidlawii S 2 and its virus MV-Lg-L 172 were (1) particles 7-19 nm in diameter, mostly located in the convex cytoplasmic fracture faces. (2) small bulges or aggregates, 13-25 nm in diameter, which occupied only limited areas of both inner and outer fracture faces of some mycoplasmas, (3) numerous tiny grains and/or spikes 2-6 nm in diameter, protruding from a finely structured background, especially in the outer concave mycoplasmal fracture faces, and (4) linear structures, most probably fibrils and thicker filaments, both in the fracture faces and around mycoplasmas and viruses and connected with them. There was a high degree of structural similarity between mycoplasmal and viral membranes: no obvious significant difference was found.
Toxic effects of both main colicin types, i.e. of porin and nuclease types, involve the direct contact of their molecules with the plasma membrane of sensitive cells. In the present study, it was tested whether this contact provokes a lateral or vertical movement of intramembrane protein particles (IMP) or a direct cleavage of the proteins. IMP were visualized by freeze-fracturing and electron microscopy on the protoplasmic fracture face (PF) of colicin-treated cells of Escherichia coli. Possible changes in distribution and in density of IMP due to treatment with colicins E1-E7 and K were followed. As a control, the bacteria were equilibrated at 0 degrees C before quenching, which caused a reversible formation of smooth areas and a decrease in the mean density of IMP on the PF. Colicins E1-E7 had no clear-cut effect on the disposition of IMP. Only colicin K decreased the IMP density, by 10% in E. coli strain 58-161 and by 17% in strain C6; the distribution of IMP remained homogeneous. Trypsin reactivation of colicin-K-inactivated bacteria was not reflected by restoration of the original density of IMP; on the contrary, it led to a further decrease, of 1-13%, in IMP density, presumably by proteolytic cleavage. Varying densities of IMP in different strains of the same bacterial species (under standard conditions) were confirmed.
- MeSH
- buněčná membrána účinky léků ultrastruktura MeSH
- elektronová mikroskopie MeSH
- Escherichia coli účinky léků MeSH
- koliciny farmakologie MeSH
- mrazové lámání MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- koliciny MeSH
Restrictive phenotype of sec 7 mutant of Saccharomyces cerevisiae was examined by freezefracture electron microscopy. In accordance with previous findings (NOVICK et al. 1981) dictyosomes and middle-size (200-600 nm) vesicles (Berkeley bodies) were found to accumulate. Dictyosomes are formed by aggregated flattened or dilated cisternae without associated secretory vesicles. After transfer to permissive conditions the dictyosomes disappear and are not detectable, just like in the wild type or in the permissive phenotype. Associated with the restrictive phenotype is an extended plasma membrane and tonoplasts with particle-free impressions.
