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PubMed1978

Membrane isolation on polylysine-coated glass beads. Asymmetry of bound membrane.

Erythrocyte membranes isolated on polylysine-coated glass beads exhibit many of the properties of the native membrane. Gel electrophoresis indicates that all major protein components of the membrane are retained during membrane isolation. The membrane integrity and accessibility of selected components was tested using non-penetrating probes. In general, membranes on beads displayed accessibility properties typical of inside-out vesicles. The accessibility of membrane acetylcholinesterase to assay reagents, as well as membrane accessibility to the actions of neuraminidase, trypsin and galactose oxidase-NaB3H4 demonstrated that the protoplasmic surface of membrane isolated on beads was exposed, while the extracellular surface was inaccessible. The differential accessibility of the membrane surfaces demonstrates the feasibility of investigating asymmetry of membranes isolated on cationic glass beads.

Acetylcholinesterase↗
PubMed1979

Alteration of human erythrocyte plasma membranes by perfringolysin O as revealed by freeze-fracture electron microscopy. Studies on Clostridium perfringens exotoxins V.

When human erythrocyte membranes were treated with perfringolysin O (Clostridium perfringens theta-toxin) and examined by electron microscopy after freeze-fracture, two ultrastructural alterations were observed in fracture faces of membrane. (1) A random aggregation of intramembranous particles was seen in the fracture face of the protoplasmic half (PF face) of all membranes treated with the toxin, even if at a low concentration (40 hemolytic units/ml). On the other hand, the aggregation in the fracture face of the exoplasmic half (EF face) was observed only in membranes treated with a high concentration (3300 hemolytic units/ml) for 2 h. (2) Round protrusions and "cavities" with 30 nm in diameter were visible in EF and PF faces of membranes treated with a high concentration, respectively. These structures were always protruded toward cytoplasmic side, but did not appear to form holes through the membrane. Ring and arc shaped structures with a dark center of 26 nm and a distinct border of 5 nm in width were observed when the toxin alone was negatively stained at a very high concentration (170,000 hemolytic units/ml). These structures were also produced in the presence of cholesterol even if the toxin concentration was low.

Bacterial Toxins↗
PubMed1981

Aggregation of intramembrane particles in erythrocyte membranes treated with diamide.

Treatment of erythrocytes with diamide (diazene dicarboxylic acid bis-(N,N-dimethylamide)) results in oxidation of sulfhydryl groups of the membrane, and cross-linking of membrane proteins into high molecular weight complexes. Concomitant freeze-etching studies show aggregation of intramembrane particles on the protoplasmic fracture face of erythrocyte ghost membranes treated with the oxidant. Furthermore, after a 3 h incubation of erythrocytes with 10 mM diamide at 37 degrees C, cellular energy levels declined to about 70% of control values. The data suggest that disulfide cross-linking of the major membrane proteins releases the apparent physical occlusion of the band 3 proteins within the interstices of the cytoskeletal shell. This results in the translational mobility of band 3 proteins which is reflected ultra-structurally in the freeze-etch images.

Adenosine Triphosphate↗
PubMed1984

Asymmetric mass distribution of (Na+ + K+)-ATPase in membranes studied by freeze-fracture-etch electron microscopy.

SDS-purified porcine kidney (Na+ + K+)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: FA, which shows a rather smooth background with many intramembranous particles, and FB, which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that FA faces are covered with many intramembranous particles, while the etched external faces (EA) are either irregularly granulated or reveal many particles half the size of intramembranous particles. FB faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (EB) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated FA convex fracture faces and irregularly granulated or smooth etched EA surfaces, indicating that the FA face is the protoplasmic fracture face and that the majority of the protein mass of the (Na+ + K+)-ATPase is located on the cytoplasmic half of the membrane.

Animals↗
PubMed1984

Cell-cycle-specific biosynthesis of the photosynthetic membrane of Rhodopseudomonas sphaeroides. Structural implications.

Structural changes association with the intracytoplasmic membrane during the cell cycle of the photosynthetic bacterium Rhodopseudomonas sphaeroides have been studied by freeze-fracture electron microscopy. The isolated intracytoplasmic membrane vesicles, chromatophores, were fused in order to obtain large fracture faces, allowing more precise measurements and statistical analysis of both intramembrane particle density and size determinations. The intramembrane particle density of the protoplasmic face (PF) of the intracytoplasmic membrane, (from 4970 to 8290/micrometers 2), was shown to be a linear function of the protein/phospholipid ratio (from 2.5 to 5.1, w/w) of the intracytoplasmic membrane. Under constant light intensity, both the average particle size and particle size distribution remained unchanged during the cell cycle. These results provide the structural basis for the earlier reported cell-cycle-specific variations in both protein/phospholipid ratio and alternation in phospholipid structure of the intracytoplasmic membrane of R. sphaeroides during photosynthetic growth. The average particle diameter in the PF face of the intracytoplasmic membrane was 8.25, 9.08 and 9.75 nm at incident light intensities of 4000, 500 and 30 ft X cd, respectively. When chromatophores were fused with small, unilamellar liposomes, the intramembrane particle density decreased as input liposome phospholipid increased, whereas the particle size remained constant and particle distribution became random.

Bacterial Chromatophores↗
PubMed1988

Altered plasma membrane ultrastructure in multidrug-resistant cells.

Multidrug resistance is mediated by P-glycoprotein, an integral plasma membrane component which is thought to function as a drug export pump. This model can explain drug resistance, but fails to account for the broader pleiotropy of the multidrug resistance phenotype. We report here a freeze-fracture study revealing increases in the densities of protoplasmic face intramembrane particles in multidrug-resistant Chinese hamster ovary (CHO) and human leukemic cells. The intramembrane particle density in a CHO cell revertant which had lost the characteristics of the multidrug resistance phenotype was indistinguishable from that of the drug-sensitive parental cell line. This demonstration of a global multidrug resistance-linked change in plasma membrane architecture may have significant implications for understanding the variety of concurrent membrane-related changes which are not easily explained by the current model for multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label-fracture and by fracture-label.

Label-fracture and fracture-label membrane immunocytochemistry are used to analyze the surface distribution, dynamics and partition on fracture of CD3 and CD4 antigens of human T lymphocytes. Redistribution of the antigens, induced by treatment at 37 degrees C with specific monoclonal antibodies, results in patching and capping of the labeling as observed in label-fractured specimens. Examination of platinum/carbon replicas of freeze-fractured plasma membranes of antibody-treated cells does not reveal recognizable domains of intramembrane particles. However, in cells where the aggregation of intramembrane particles is induced by incubation with glycerol, colloidal gold-labeled CD3 and CD4 molecules are seen confined to particulate domains of the membrane. Therefore, the lack of visible aggregation of intramembrane particles in patched or capped regions of the membrane implies that migration of CD3 and CD4 antigens with concentration in domains of the membrane is achieved contemporaneously with export of other non-capped integral membrane proteins from the same regions, in a process of diffusional equilibrium. Examination of fracture-labeled specimens shows that CD4 molecules partition on fracture with the inner protoplasmic face of the plasma membrane. This partition illustrates the transmembrane attitude of the antigen molecule and is a probable consequence of interaction of the protein with other components of the membrane or with the cytoskeleton.

Antibodies, Monoclonal↗
PubMed1990

Ultrastructural alterations in plasma membranes from drug-resistant P388 murine leukemia cells.

Freeze-fracture studies of daunomycin-sensitive and daunomycin-resistant P388 cell lines, reveal a significant increase in the numerical density of intramembrane particles at both, the protoplasmic and the exoplasmic leaflets of the plasma membrane from the drug-resistant cells. Such change in plasma membrane architecture is not accompanied by overexpression of P-glycoproteins. Furthermore, drug-sensitive cells exhibited an increased number of exo-endocytotic images when compared to drug-resistant cells. Our observations suggest that there are global changes in the structural organization of the plasma membrane, which are related to the acquisition of the cellular drug-resistant phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1977

Membrane associated particles of the presynaptic active zone in rat spinal cord. A morphometric analysis.

The distribution of membrane-associated particles in the presynaptic membrane was investigated in the spinal cord of unanesthetized and anesthetized rats by freeze etching electron microscopy. Both 'external' face (EF) and 'protoplasmic' face (PF) were examined. Particles were classified according to size in two categories: small particles (diameter 5.0-8.7 nm) and large particles (diameter 8.7-13.7 nm). The presynaptic region was subdivided into an active and a surrounding zone, depending on specific ultrastructural criteria. The density of large particles in the PF was found to be significantly higher in the active as compared with the surrounding zone in both unanesthetized and anesthetized rats. Thus, the presence of large particles represents an important feature of the active zone. Considerably more large particles were found in the waking than in the barbiturized state. This difference is paralleled by a vast increase of vesicle attachment sites in the presynaptic membrane of unanesthetized animals reported by Streit et al. and confirmed in the present study. It is suggested that the two phenomena could be interrelated and that the large particles may represent calcium channels and thus provide the morphological substrate for the mechanism of excitation-secretion coupling.

Animals↗
PubMed1978

Intramembranous particles at the nodes of Ranvier of the cat spinal cord: a morphometric study.

Size and distribution of intramembranous particles at nodes of Ranvier of the cat spinal cord were investigated by the freeze-etching technique and compared with those at the internodal axon. The particles are larger (up to 20 nm) at the nodal than at the internodal segment (up to 13 nm), and these large particles are more densely packed in the nodal (400 per sq micrometer) than in the internodal E (external) face (4 per sq micrometer). The nodal E face reveals a much denser overall population (1200--1300 per sq micrometer) of particles than the internodal E face (100--200 per sq micrometer), while at the P (protoplasmic) face the particle density is similar in nodal and internodal segments (1200--1600 per sq micrometer). It is suggested that the large nodal particles may be related to the mechanism of nerve excitation.

Animals↗
PubMed1979

Ultrastructural effects of 4-aminopyridine on the presynaptic membrane in the rat spinal cord.

The effects of 4-aminopyridine (4-AP, 1 mg/kg, i.v.) on the ultrastructure of synapses in the ventral horn of the spinal cord were examined in rats anesthetized with 50 and 100 mg/kg, i.p., pentobarbital sodium and alpha-chloralose respectively. The presynaptic membrane of the 4-AP-treated animals, in both S- and F-type synapses, was wrinkled and gave frequently rise to omega-shaped profiles. However, it was not prominently lifted in comparison with the controls. In both extracellular (EF) and protoplasmic fracture faces (PF) of freeze-etched replicas, the frequency of the presynaptic membrane modulations (PMM) per unit area rose significantly under the influence of 4-AP. The ratio of open (crater-like) to total PMM in EF increased almost twice under the experimental condition. No differential 4-AP effects were seen between pentobarbital- and chloralose-treated animals. The morphological evidence is consistent with electrophysiological data showing that 4-AP facilitates the transmitter release at synaptic endings.

Aminopyrine↗
PubMed1979

Increase of large intramembranous particles in the presynaptic active zone after administration of 4-aminopyridine.

The effects of 4-aminopyridine (4-AP, 1 mg/kg i.v.) on the distribution of intramembranous particles in spinal motoneuronal synapses was examined in anesthetized (pentobarbital sodium, 50 mg/kg, i.p.) rats by freeze-etching electron microscopy. The particles were classified according to their size into small (4.2--10.5 nm in diameter) and large (10.5--18.9 nm in diameter). The active zone of the presynaptic membrane was identified on the basis of ultrastructural criteria. The large intramembranous particles in both protoplasmic (PF) and external fracture faces (EF) were aggregated more densely in the active zone as compared with its surroundings both in the control and 4-AP-treated group (P less than 0.001). The density of large particles in the PF rose from 300/sq.micron under the influence of 4-AP (P less than 0.001). The increased frequency of vesicle attachment sites at the presynaptic membrane (from 115/sq.micron in barbiturized controls to 150/sq.micron in 4-AP treated animals) seemed to be positively correlated with the increasing density of large particles. Based on these findings it is suggested that the large intramembranous particles of the presynaptic membrane may participate in the control of calcium fluxes which are essential to the transmitter release by exocytosis.

Aminopyridines↗
PubMed1981

Dye-coupling between glial cells in the guinea pig neocortical slice.

Physiologically identified glial cells in guinea pig neocortical slices were injected with the low molecular weight, fluorescent dye Lucifer yellow CH. The stained aggregates which resulted consisted of one brightly stained, central cell surrounded by numerous lightly stained cells. The central cell had well defined feathery processes and resembled a protoplasmic astrocyte. The surrounding cells appeared also to be glial cells but lacked sufficient detail to be further categorized. This first demonstration of dye-coupling between neocortical glial cells strongly suggests that these cells are connected together via low resistance junctions capable of passing ionic current as well as dye.

Animals↗
PubMed1983

Immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis.

Spinal cord sections from Lewis rats with acute experimental allergic encephalomyelitis (EAE) showed greatly increased staining of astrocytes when stained immunocytochemically for glial fibrillary acidic protein (GFAP). Fibrous processes in white matter were heavily stained early in the course of the disease when paralysis was first evident (10-12 days after injection of guinea pig spinal cord myelin), then protoplasmic astrocytes were stained in the gray matter and became more heavily stained at 20 days post-injection. The stained astrocytes were evenly distributed throughout the tissue, and did not correspond to the sites of the lesions. Spinal cord slices of control and EAE rats were incubated with [3H]amino acids, then cytoskeletal proteins were prepared in an enriched fraction, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands counted for radioactivity. In the EAE rat all cytoskeletal proteins, including the neurofilaments, vimentin, microtubules, GFAP and actin, showed increased uptake of radioactive amino acids. Immunoprecipitation of GFAP with specific antiserum showed increased radioactivity in the complex beginning at day 10 when cellular infiltration was beginning in the EAE animals. As the disease became acute, the radioactivity in the immunoprecipitated GFAP increased, in some cases to very high levels, then by day 18 when recovery was underway, the radioactivity had fallen to normal levels. Possible agents causing metabolic activation of protein synthesis in EAE animals include stimulating substances elaborated by infiltrating lymphoid cells, and the generalized edema accompanying the demyelinative condition. The activation of GFAP protein staining and metabolism in EAE might serve as a model for the activated growth of astrocyte processes which cause the severe gliosis seen in multiple sclerosis.

Animals↗
PubMed1987

Astrocytes in rat fetal cerebral cortical homografts following implantation into adult rat spinal cord.

The present experiment examines astrocytes in fetal cerebral cortical homografts to adult rat spinal cords. The purpose of this study is to determine if astrocytes are structurally organized within the graft. Also, the presence or absence of astrogliosis may be an indicator of the metabolic status of the graft. Embryonic cerebral cortex was taken at 14 days gestational age and transplanted into adult spinal cord at the level of the sixth thoracic vertebra. The homografts were examined at the light and electron microscopic levels from 7 days postimplantation (PI) to 6 months PI with glial fibrillary acidic protein antiserum which is a specific immunohistochemical marker for astrocytes. At 7 days PI, immunoreactive astrocytes were present only at the periphery of the graft and appeared to be associated with blood vessels. By 30 days PI, normal protoplasmic astrocytes were present throughout the graft. No hypertrophied astrocytes are present at 30 days PI, but the numerical density of astrocytes is greater than that of normal cerebral cortical gray matter. Fibrous astrocytes are present in the periphery of the implant and many of these astrocytes extended their processes between the host and the graft. Occasional glial scarring is observed between the gray matter of the host and graft, but generally no glial scar occurred in the interface between the graft and the host gray matter. By 45 days PI, hypertrophied astrocytes can be seen in the graft, but are confined in this age group to perivascular regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Modification of the Na+,K+-pump of glial cells within cobalt-induced epileptogenic cortex of rat.

The characteristics of a glial Na+,K+-pump dependent on extracellular K+ within epileptogenic cortex were studied electrophysiologically, biochemically and histochemically in vitro using slices from cobalt-induced epileptogenic cortex of rat. When the extracellular K+ concentration ([K+]o) was varied between 4 and 40 mM, the mean slope of membrane potential plotted against [K+]o was about 57 mV in glia from the normal cortex (tissue A) and about 44 mV in glia from the epileptogenic cortex (tissue B); whereas no significant difference in the resting membrane potential of these tissues was observed. In glia from tissue B, a marked transient hyperpolarization above control level was caused by replacement of elevated [K+]o with the normal medium. Ouabain abolished these phenomena observed in glia from tissue B, but had no effect on the membrane potential during normal [K+]o. Reduction of extracellular Na+, Ca2+ and Cl- did not significantly affect the membrane potential of glia from either tissue. In tissue A, the cells marked by intracellular injection of horseradish peroxidase after intracellular recording were protoplasmic astrocytes; in tissue B, fibrous astrocytes with abnormal processes predominated. K+-dependent stimulation of Na+,K+-ATPase activity of the astrocyte-enriched fraction and its membrane preparation from tissue B was much larger than that from tissue A. A certain amount of the reaction product of K+-pNPPase activity was seen on glial plasma membrane within tissue B but not on that from tissue A. The above findings suggest that a glial Na+,K+-pump within actively firing epileptogenic cortex may be modified to increase in its activity.

Animals↗
PubMed1988

Quantitative studies on proliferative changes of reactive astrocytes in mouse cerebral cortex.

Cell number and proliferation of reactive astrocytes were studied quantitatively in the stabbed cerebral cortex of adult mice, using immunohistochemistry for glial fibrillary acidic protein (GFAP) and [3H]thymidine autoradiography. GFAP-positive astrocytes increased in cell number gradually from 24 to 96 h after stabbing, and their immunoreactivity became intense. The maximum number of GFAP-positive cells was about 4.5 times normal in the layers II-VI of the cortex, whereas it was only 1.5 times normal in the layer I (molecular layer). In contrast to the gradual increase in cell number, no GFAP-positive astrocytes were labeled with [3H]thymidine prior to 48 h after stabbing, in either the layer I or the layers II-VI. Then 3-5% of them were labeled at 72 and 96 h, but very few again after 6 days. By injecting [3H]thymidine successively for 6 days after stabbing, only 17% of GFAP-positive astrocytes of the layer I or the layers II-VI were labeled. These results reveal that, in the cortical layers II-VI, many GFAP-negative source cells initially express much more GFAP-antigen without proliferation and change into GFAP-positive reactive astrocytes. Proliferation of reactive astrocytes is not the major factor for the marked increase in number of them. The cortical layer I would have few GFAP-negative source cells for reactive astrocytes. These source cells may be protoplasmic astrocytes.

Animals↗
PubMed1989

Degeneration of neurons in the thalamic reticular nucleus following transient ischemia due to raised intracranial pressure: excitotoxic degeneration mediated via non-NMDA receptors?

Transient global ischemia was produced in rats by cisternal fluid infusion, producing a negative cerebral perfusion pressure by elevating the intracranial pressure (ICP) 25-50 mm Hg above mean arterial pressure (MAP). Animals were allowed to survive for 2-7 days following a transient ischemic episode of 5-30 min. The brains were examined for signs of ischemic degeneration in Nissl-stained sections and adjacent sections reacted with antisera against glial fibrillary acidic protein (GFAP) or aspartate aminotransferase (AAT). Neurons in the thalamic reticular nucleus (RT), a pure population of gamma-aminobutyric acid (GABA)ergic neurons which project their axons to thalamic relay nuclei, were found to have the lowest threshold for degeneration in this model, consistently undergoing degeneration under conditions which completely spared the hippocampal CA1 from degeneration. Whereas it took up to 30 min of complete ischemia to produce degeneration of CA1 neurons when ICP was raised using room temperature infusion fluids, 15 min of ischemia under these conditions was sufficient to produce extensive degeneration of neurons in the entire ventral 3/4 of the RT. Prolonged (greater than 25 min) episodes of partial ischemia (ICP less than or equal to MAP) were also sufficient to produce massive degeneration of RT neurons. The lesion in the RT was most clearly evident in sections reacted with antisera to GFAP, labeling intensely reactive protoplasmic astrocytes within the regions of the RT where neuronal degeneration had occurred. Neuronal loss and accompanying proliferation of microglial cells were evident in Nissl-stained sections but the extent of the neuronal loss was most clearly obvious in sections reacted with an antisera to AAT, an enzyme present in detectable quantities in GABAergic neurons. Pretreatment with the non-competitive NMDA antagonist MK-801 at doses sufficient to completely prevent massive degeneration of the hippocampal CA1 failed to prevent the degeneration of RT neurons, suggesting that if RT degeneration involves an excitotoxic process it acts through non-NMDA receptors.

Animals↗