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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 61 (1993), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The major postsynaptic density protein, proposed to be a calcium/calmodulin-dependent protein kinase, becomes phosphorylated when a postsynaptic density preparation from rat cerebral cortex is incubated in medium containing calcium and calmodulin. Upon longer incubation, however, the level of phosphorylation declines, suggesting the presence of a phosphatase activity. When Microcystin-LR, a phosphatase inhibitor, is included in the phosphorylation medium, the decline in phosphorylation is prevented and a higher maximal level of phosphorylation can be achieved. Under these conditions, the maximal phosphorylation of major postsynaptic density protein is accompanied by a nearly complete shift in its electrophoretic mobility from 50 kDa to 54 kDa, similar to that described for the a subunit of the soluble calcium/calmodulin-dependent protein kinase II. Of the four major groups of serine/threonine protein phosphatases, the enzyme responsible for the dephosphorylation of major postsynaptic density protein is neither type 2C, which is insensitive to Microcystin-LR, nor type 2B, which is calcium-dependent. As Microcystin-LR is much more potent than okadaic acid in inhibiting the dephosphorylation of major postsynaptic density protein, it is likely that the postsynaptic density-associated phosphatase is a type 1. The above results indicate that the relatively low level of phosphorylation of the major postsynaptic density protein observed in preparations containing postsynaptic densities is not due to a difference between the cytoplasmic and postsynaptic density-associated calcium/calmodulin-dependent kinases as previously proposed, but to a phosphatase activity, presumably belonging to the type 1 group.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: A widely used method for the preparation of postsynaptic density (PSD) fractions consists of treatment of synaptosomal membranes with Triton X-100 and further purification by density gradient centrifugation. In the present study, the purity of this preparation was assessed by electron microscopic analysis. Thin-section and rotary shadow immuno-electron microscopy of the Triton X-100-derived PSD fraction shows many PSD-95-positive structures that resemble in situ PSDs in shape and size. However, the fraction also includes contaminants such as CaMKII clusters, spectrin filaments and neurofilaments. We used magnetic beads coated with an antibody against PSD-95 to further purify PSD-95-containing complexes from the Triton-derived PSD fraction. Biochemical analysis of the affinity-purified material shows a substantial reduction in the astrocytic marker glial fibrillary acidic protein and electron microscopic analysis shows mostly individual PSDs attached to magnetic beads. This preparation was used to assess the association of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors with the PSD-95-containing complex. AMPA receptors are demonstrated by immunoblotting to be present in the complex, although they do not co-purify exclusively with PSD-95, suggesting the existence of two pools of receptors, one associated with the PSD-95 scaffold and the other not. Of the AMPA receptor-anchoring proteins tested, SAP-97 is present in the affinity-purified preparation whereas GRIP is found only in trace amounts. These results imply that a subpopulation of AMPA receptors is anchored to the PSD-95-containing scaffold through interaction of GluR1 with SAP-97.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 483 (1986), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 296 (1982), S. 464-466 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Tight junction strands have been assumed to consist of a row of intramembrane particles representing integral membrane proteins which on fixation (glutaraldehyde 1-5%) and cryo-protection (glycerol 20-30%) form the continuous fibrils observed in standard freeze-fracture replicas5'7'12'16. This ...
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Journal of neurocytology 14 (1985), S. 943-960 
    ISSN: 1573-7381
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The structure and organization of axons in the cervical connective of wild-typeDrosophila fruit flies were examined in anticipation of studies of various neurological mutants. Dissected flies were rapid-frozen from the living state against a copper block cooled with liquid helium, freeze-substituted, and prepared for electron microscopic examination of thin sections. These cryotechniques showed new details of the structure of cell organelles and cytoplasm inDrosophila axons. The cytoplasmic matrix of axons and glia consists of a material with a fine granular texture enmeshed in a three-dimensional meshwork of short, fine filaments which vary in shape, size and electron density. No neurofilaments are present, but bundles of microtubules are interwoven into the filamentous matrix of the axoplasm. The round wall of microtubules (27 nm overall diameter) is composed of twelve cylindrical protofilaments with a typical substructural periodicity. Mitochondria frequently make contact with microtubules in both axons and glial processes. A thin layer of electron-dense filamentous matrix, which appears to be an axonal basal lamina, contacts most of the axonal exoplasmic surface, especially that of axons where they are surrounded by processes of glial cells, but is scant wherever single axons are contiguous. Thus, an axonal basal lamina occupies the constricted spaces around axons, where extracellular K+ accumulates during neural activity.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Journal of neurocytology 10 (1981), S. 183-199 
    ISSN: 1573-7381
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The outer plexiform layer (OPL) of the developing chick retina from 11 embryonic days to 11/2 weeks posthatching has been studied by freeze-fracture to characterize changes in the membrane structure of photoreceptor terminals during synaptogenesis. At early stages, the undifferentiated photoreceptor synaptic base is characterized by a sparse distribution of intramembrane particles on the inner leaflet (P-face). Later, as the synaptic base begins to differentiate by extending filopodia into the OPL, numerous small aggregates of large particles appear between and on filopodial surfaces. Many of the aggregates occupy crater-like depressions, which are seen in cross-fractures through the underlying cytoplasm to be associated with vesicular invaginations of the presynaptic membrane. Corresponding thin sections through these regions at this time reveal immature arciform densities and coated vesicles fusing with the presynaptic membrane adjacent to these densities. At later stages, many of the particle aggregates on the photoreceptor membrane appear to have coalesced into longer arrays overlying ridges surrounded by numerous vesicle fusion sites. These intramembrane changes correlate with the formation of the mature arciform density-synaptic ribbon specialization in the photoreceptor presynaptic terminal and with physiological maturation of the chick retina.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 291-300 
    ISSN: 0886-1544
    Keywords: endoplasmic reticulum ; DiOC6(3) ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Relationships among the endoplasmic reticulum (ER), microtubules, and bead movements on the cell surface were investigated in the thin peripheral region of A6 cells, a frog kidney cell line. ER tubules were often aligned with microtubules, as shown by double-labeling with DiOC6(3) and anti-tubulin in fixed cells. In living cells stained with DiOC6(3) and observed in time lapse, there were frequent extensions, but few retractions, of ER tubules. In addition, there was a steady retrograde (towards the cell center) movement of all of the ER at ∼0.3 μm/min. Since microtubules are often aligned with the ER, microtubules must also be moving retrogradely. By simultaneous imaging, it was found that the ER moves retrogradely at the same rate as aminated latex beads on the cell surface. This indicates that the mechanisms for ER and bead movement are closely related. Cytochalasin B stopped bead and ER movement in most of the cells, providing evidence that actin is involved in both retrograde movements. The ER retracted towards the cell center in nocodazole while both ER and microtubules retracted in taxol. Time lapse observations showed that for both drugs, the retraction of the ER is the result of retrograde movement in the absence of new ER extensions. Presumably, ER extensions do not occur in nocodazole because of the absence of microtubules, and do not occur in taxol because taxol-stabilized microtubules move retrogradely and there is no polymerization of new microtubule tracks for ER elongation. © 1994 Wiley-Liss, Inc.This Article is a US Government work and, as such, is in the public domain in the United States of America.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    The @Anatomical Record 213 (1985), S. 7-15 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Intercellular junctions, in identified in freeze-fracture by narrowing of the intercellular gap and codistribution of P-face membrane particles, proliferate during incubation of excised rat prostate tissue in 30% glycerol solution. These junctions fulfill the criteria used to identify gap junctions in freeze-fracture replicas with respect to the size and uniformity of their component particles, the codistribution of the particle aggregates on P-faces of adjacent membranes, and the narrowing of the extracellular cleft at the junction. These gap-junction-like structures form on the lateral surfaces of epithelial cells, where they are normally scarce, within minutes after exposure to glycerol. Glycerol-induced junction formation is not blocked by DNP, a metabolic uncoupler, or by cycloheximide, a protein synthesis inhibitor. Newly formed junctions occur initially in clusters and the number per cluster decreases as individual junctions become larger with longer periods of incubation, suggesting that the clusters coalesce. The structural changes that precede and accompany the formation of these junctions at early times of incubation are comparable to the changes reported to precede the formation of gap junctions accompanying hormonal treatment, development, and other means of natural induction.
    Additional Material: 14 Ill.
    Type of Medium: Electronic Resource
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