SEARCH · PubMed Health

Results for “Freeze Fracturing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources

PubMed1989

Perturbation of the sarcolemmal membrane in isoproterenol-induced myocardial injury of the rat. Permeability and freeze-fracture studies in vivo and in vitro.

The mechanism whereby cardiotoxic doses of isoproterenol (ISO) induces early permeability alteration of the sarcolemmal membrane is unknown; both beta-receptor overstimulation and direct toxic effect of ISO oxidation products have been implicated. There has been no morphologic observation, furthermore, on the structural basis of permeability alteration during this process. The purpose of the present study was to compare the morphology of cardiocyte injury induced by ISO and oxidized ISO (ISO-O2) and to visualize perturbation of the sarcolemma correlating with the leaky membrane. The authors studied the left ventricular myocardium of rats 10 and 60 minutes after subcutaneous administration of 85 mg/kg ISO and isolated perfused rat hearts exposed for 10 minutes either to ISO or ISO-O2 in a dose of 100 mg/l (10(-4) M) to determine the permeability of the sarcolemmal membrane using the extracellular diffusion tracer horseradish peroxidase (HRP) by light and thin section electron microscopy, the morphology of the sarcolemmal membrane by means of freeze-fracture electron microscopy, and the density of intramembrane particles (IMP) in the sarcolemmal membrane by planimetry using freeze-fracture electron microscopy. In in vivo rat hearts both 10 and 60 minutes after ISO and in vitro (isolated perfused) rat hearts exposed to either ISO or ISO-O2 for 10 minutes, HRP labeled the sarcoplasm of focally located cardiocytes implicating leakiness of the sarcolemmal membrane. HRP positive cardiocytes (with the exception of the in vivo 10 minute group) showed characteristic features of contraction band necrosis (both on light and thin-section electron microscopy) in all groups. Freeze-fracture electron microscopy of sarcolemmal protoplasmic (P) membrane faces revealed two populations of cardiocytes in all groups. P-membrane faces in one population of cardiocytes appeared as in the control. In the other population of cardiocytes, P-membrane faces showed irregular tears. Planimetry demonstrated a significant decrease of IMP numerical densities in P-membrane faces with tears in the in vivo 10 minute group and both with or without tears in the in vivo 60 minutes group and the in vitro groups compared with the control values. Furthermore, with the exception of the 10 minute in vivo group, IMP densities significantly decreased in sarcolemmal membranes with tears compared with those without tears in all experimental groups. These observations are consistent with the view that catecholamine induced myocardial injury is, at least partly, related to the direct toxic effect of catecholamine oxidation products on the sarcolemmal membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1983

Lack of orthogonal particle assemblies and presence of tight junctions in astrocytes of the goldfish (Carassius auratus). A freeze-fracture study.

The cytoplasmic membranes of astrocytes in the optic nerve of the goldfish were investigated by means of freeze-fracture techniques. Astrocytes of normal and regenerating optic nerves did not differ in the fine structure of plasma membranes. Emphasis is placed on the following results: Astrocytic membranes of fish do not reveal the orthogonal particle assemblies that are believed to be generally characteristic for astrocytes in the white matter. Astrocytes reveal extensive membrane areas occupied by tight junctions, which to date have not been described as characteristic astrocytic structures. These junctions are astro-astrocytic and are frequently intercalated by gap junctions. Desmosomes are another characteristic type of astro-astrocytic junction. By means of freeze-fracture replicas it can be demonstrated that they occur in more or less close association with tight and gap junctions. Caveolae are also seen in the astrocytic membranes of fish: their density and distribution show distinct variations. Caveolae occur at the interface between astrocytes and the interstitial space, between astrocytes and myelin sheaths, and in astrocytic processes. It is suggested that the differences between the astrocytic membranes of fish and mammals reflect different physiological functions. They are discussed in relation to the problem of neuronal-glial interrelationships and the behavior of astrocytes during fiber regeneration in the CNS.

Animals↗

PubMed1980

Effects of glutaraldehyde and glycerol on freeze-fractured Escherichia coli.

Glutaraldehyde and glycerol are widely used in the freeze-fracture technique as sample pretreatments before rapid freezing. However, they can both introduce relevant structural changes and influence the visualization of the fracture faces and surfaces of membranes. A comparison of the results obtained on E. coli cells differently pretreated with glutaraldehyde and glycerol is presented. In particular the effect on the distribution and density of the intramembranous particles (IMP) is pointed out. Glycerol treatment at 310 K introduces an IMP redistribution, outlined by the appearance of several smooth areas on the fracture faces of the cytoplasmic membrane, which is prevented by glutaraldehyde prefixation at the same temperature. On the other hand, glutaraldehyde treatment at 310 K following glycerol incubation results in the disappearance of the smooth areas, suggesting a substantial change in the IMP distribution caused by the fixative. Cells shifted down to 277 K and treated with glycerol at this temperature before quick-freezing, present on the convex fracture face of the cytoplasmic membrane large smooth areas resulting from a lipid transition while the smaller areas observed at 310 K are not detectable. Glutaraldehyde treatment at 277 K seems also to be responsible for a redistribution of IMP since poorly delimited large smooth areas, containing several IMP, can be observed. In this paper the results of a statistical analysis are also reported, showing that the IMP density can be strongly influenced by pretreatments.

Aldehydes↗

PubMed1976

The sarcolemma of Aplysia smooth muscle in freeze-fracture preparations.

Smooth muscle cells in the sheath covering the visceral ganglion of Aplysia californica were examined with the techniques of freeze-fracture and conventional electron microscopy. The sarcolemma of these muscle cell invaginates to form myriad caveolae that have an intrinsic marker within their membrane. This intrinsic structure of the caveolar membrane is revealed by freeze-fracture and consists of rows of large particles in the outer half and matching grooves on the complementary inner half of the membrane. In thin plastic sections, parallel striations or shelves within the caveolar membrane appear to be the equivalent of the particles and grooves of the fractured membrane. Physical fixation of some specimens by rapid freezing in super-cooled liquid nitrogen or in liquid helium suggests that in their natural state, the caveolar ostia are not uniform in size and that at any given moment a number of caveolae are flattened. When segments of the connective nerves which link the visceral ganglion to the cephalic ganglia are stretched in vitro two to three times their in situ length, the caveolae lose their invaginated shape and are fully exposed to the extracellular space. The caveolar membrane, so stretched, is pulled into the line of fracture with the result that the large particles rather than the ostia appear on the cleaved surface. This flattening of the caveolae is reversible and suggests that they might serve as miniature stretch-receptors within the membrane of the smooth muscle cells. The caveolae are accessible to extracellular horseradish peroxidase but do not appear to pinocytose the protein.

Animals↗

PubMed1990

Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins.

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.

Animals↗

PubMed1977

Freeze-fracture study of the carotid body.

Replicas of freeze-fractured rat carotid body chief (Type I) cells reveal the normal complement of cytoplasmic organelles. En face fractures of the chief cell plasmalemma exhibit occasional sites of fusion of the amine-containing vesicles which characterize the carotid body. The endothelial cells lining the capillary network of the carotid body display numerous pinocytotic vesicles often oriented in linear arrays, and occasional fenestrae. The endothelial cells are joined together by tight junctions which appear as grooves (B face) forming a continuous network over the membrane.

Animals↗

PubMed1975

A freeze-fracture study of adult Calliphora salivary glands.

A freeze-fracture study of adult Calliphora salivary glands has revealed a high density (approx. 4500/mu2) of intramembraneous particles (80-110 A) in both the apical and basal membranes. Most of the particles were associated with the A face. The density of the stalked surface particles which coat the cytoplasmic surface of the apical membrane. The possible significance of these particles in ion transport is discussed.

Animals↗

PubMed1978

The direct measurement of temperature changes within freeze-fracture specimens during rapid quenching in liquid coolants.

We have evaluated the cooling rates of specimens mounted in a variety of freeze-fracture holders when plunged into a series of liquid coolants. these rates were measured using miniature thermocouples placed within the mounted specimens. The most rapid cooling rates were obtained using propane at 83 K as the coolant. When mounted on a newly devised 'copper sandwich' holder, specimen cooling rates in excess of 4500 K/s have been recorded. A simple guillotine-like device for quenching freeze-fracture specimens under reproducible conditions is presented.

Chlorofluorocarbons, Methane↗

PubMed1990

Freeze-fracture and immunomorphological analysis of spiral ganglion cells.

Freeze-fracture analysis of adult spiral ganglion cells of CBA/CBA mice revealed two types of membrane specializations. Most cells (type I) had a smooth surface and were surrounded by Schwann cells. Type II spiral ganglion cells showed numerous membrane specializations with well-delineated indentations similar to those previously found on hair cells adjacent to afferent and efferent nerve endings. Immunomorphological analysis (using well-defined monoclonal antibodies directed against different subclasses of intermediate filament proteins) revealed a unique co-expression of neurofilaments, vimentin and cytokeratins in spiral ganglion cells of 8- to 22-week human fetuses.

Animals↗

PubMed1982

Quantitative analysis of intramembrane particle (IMP) distribution on biomembranes after freeze-fracture preparation by a computer-based technique.

An improved method for the quantitative analysis of intramembrane particles (IMPs) on freeze-fracture faces of biomembranes is described. The analysis provides, first, the number of IMPs per 1 micrometer2, second, the coefficient of dispersion (CD) of unweighted and weighted (i.e. the finite size is taken into consideration) IMP distribution, and, third, the diameters of IMPs. Additional data for further statistical evaluation are also derived. These data make it possible to numerically describe and characterize fracture faces of a membrane in a defined physiological state. They allow for comparison of normal membranes with experimentally induced alterations of the same specimen in other experiments, and for observation of differences between normal and malignant cells. Furthermore, such an analysis provides the basis for a dynamic interpretation of biomembrane components seen on the static freeze-fracture micrographs. With this numerical analysis, descriptive terms for IMP distribution such as 'random', 'aggregated' or 'clustered', which cannot be evaluated or verified, are avoided. The method, based on the Poisson statistic which provides a means for the description of different particle density distributions, is described. The validity of the method is demonstrated on artificially generated pictures and by an analysis of erythrocyte membranes before and after spectrin depletion.

Computers↗

PubMed1978

A freeze-fracture study of membrane events during neurohypophysial secretion.

Freeze-fracture was used to study the membrane events taking place during neurosecretory granule discharge (exocytosis) and subsequent membrane internalization (endocytosis) in axons of neurohypophyses from control and water-deprived rats. En face views of the cytoplasmic leaflet (P face) of the split axolemma reveal circular depressions that represent the secretory granule membranes fused with the plasma membrane during exocytosis. These depressions often contain granule core material in the process of extrusion into the extracellular space. The membrane surrounding some of the exocytotic openings shows a decreased number of intramembrane particles (mean diameter, 8 nm) which are elsewhere more numerous and evenly distrubuted on the fracture face. Endocytotic sites appear as smaller plasma membrane invaginations, with associated intramembrane particles. Moreover, such invaginations often contain large particles (mean diameter, 12 nm) that appear as clusters on en face views of the membrane leaflet. Quantitative analysis indicates that the number of exocytotic images increases significantly in glands from water-deprived rats. Concomitantly, the number of endocytotic figures per unit area of membrane is raised as is the number of clusters of large particles. The observations demonstrate that, in the neurohypophysis, it is possible to distinguish exocytosis morphologically from endocytosis and that the two events can be assessed quantitatively.

Animals↗

PubMed1978

Freeze-fracture of membrane fusions in phagocytosing polymorphonuclear leukocytes.

Freeze-fracture of rabbit polymorphonuclear leukocytes in the process of phagocytosing yeast cells shows changes in storage granule and phagosome membranes essentially similar to those described in mammalian secretroy cells during exocytosis. These changes consist of the clearing of intramembrane particle from limited zones of the fusing granule and phagosome membranes. After the completion of fusion, which leads to the incorporation of storage granule membrane into the phagosome membrane, particle-free patches are no longer visible, but the phagosome membrane contains some loose aggregates of particles. These data suggest that intracellular membrane fusion in polymorphonuclear leukocytes occurs through interaction of protein-depleted areas of the involved membranes.

Animals↗

PubMed1983

Reinforcement and protection with polystyrene of freeze-fracture replicas during thawing and digestion of tissue.

An improved method for reinforcing freeze-fracture replicas, which is especially suitable for tough tissues, is described. For this purpose, polystyrene dissolved in chloroform is used to produce a solid protecting layer of plastic on top of the replica, enabling it to withstand the stresses associated with the thawing and digestion of the tissue with strong acids. The method results in production of large clean replicas from tissues such as skin or peripheral nerve which are difficult to process. The method can also be used profitably for reinforcing other softer and more homogeneous tissues.

Axons↗

PubMed1981

Digestive system membranes: freeze-fracture evidence for differentiation and flow in Paramecium.

Freeze-fractured membranes of digestive vacuoles of randomly feeding Paramecium caudatum exhibit dramatic differences in intramembrane particle (IMP) number and distribution on both E- and P-fracture faces. By pulse-feeding latex spheres to cells we have demonstrated that these differences are related to the age of the digestive vacuoles, and that the membranes of such vacuoles undergo a specific sequence of changes during the digestive cycle. Young digestive vacuoles (DV-I; less than or equal to 6 min), nascent vacuoles still connected to the cytopharynx, and discoidal vesicles, from which vacuole membrane is derived, all have a highly particulate E face and a less particulate P face. As early as 3 min after feeding, a second category of digestive vacuoles (DV-II) can be recognized, which are both considerably smaller in diameter and lack particles on their E face. These findings suggest that the endocytic removal of DV-I membrane material associated with the formation of DV-II vacuoles involves a concomitant and selective removal of E-face particles, as essentially no changes are seen in the density of P-face particles on the two types of vacuoles. Beginning at 10 min the first DV-III vacuoles are encountered. These are both larger than the DV-II vacuoles and possess very prominent E-face particles, which resemble those on the E face of the numerous lysosomes bordering the digestive vacuoles. DV-III vacuoles also exhibit a substantial increase in P-face particles. These membrane changes closely parallel, and are probably correlated with, the physiological events occurring within the vacuole lumen: concentration of food, killing of prey, and digestion. Calculations of the amount of membrane removed from DV-I to form DV-II and of the increase in membrane surface area during the transition from DV-II to DV-III indicate that as much as 90% of the initial phagosome (DV-I) membrane can be removed before digestion begins. The enlargment of DV-II must be caused by fusion with adjacent lysosomes which also contribute the new populations of IMPs to the DV-III membrane. The appearance of numerous endocytic structures on older DV-III vacuoles suggests that membrane is retrieved from DV-III before defecation.

Animals↗

PubMed1983

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol.

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin-binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze-fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment.

Animals↗

PubMed1984

New observations on the ultrastructure of mammalian conducting airway epithelium: application of liquid propane freezing, deep etching, and rotary shadowing techniques to freeze-fracture.

Freshly isolated, viable hamster tracheal epithelium was rapidly frozen in a jet of liquid nitrogen-cooled propane followed by freeze-fracture, deep etching, and rotary shadowing. Examination of the replicas by transmission electron microscopy revealed characteristic features of both ciliated and nonciliated cells, profiles of tight junctional complexes, and two distinct types of membrane particle complexes. The findings of this study suggest that rapid freezing of viable biological specimens coupled with freeze-fracture, deep etching, and rotary shadowing may provide a useful approach to achieving some additional perspectives of cell structure and function. Since the experimental protocol avoided any use of chemical fixation and processing procedures generally employed in electron microscopy, this study also validates for airway epithelium the details of fine structure observed using more conventional ultrastructural methods.

Animals↗

PubMed1981

Ultrastructural observation of neonatal lungs with transmission electron microscope and freeze-fracture replication.

Nine neonatal and one child lungs in various conditions were examined with electron microscopy combined with thin section and freeze-fracture replication techniques in order to study mainly ultrastructural changes of lamellar inclusion bodies of type II alveolar cells. Lungs from neonates with respiratory distress syndrome and with bronchopneumonia showed occasional lamellar inclusions that were partially amorphous in thin section and granular and fragmentarily linear in freeze-fracture preparation. This change was considered to represent focal micelle formation of phospholipid lamellae. No specific morphological changes were noted in lungs of respiratory distress syndrome. In contrast, lungs from thanatophoric dwarfism showed ripple appearance of inclusion lamellae. This was considered to possibly represent unusual phospholipid compounds forming lamellae. Other interesting findings were also described.

Child, Preschool↗

PubMed2000

Early myelin breakdown following sural nerve crush: a freeze-fracture study.

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.

Animals↗