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  • 1
    ISSN: 0886-1544
    Keywords: NBD-phallacidin ; actin ; ocular tissues ; wound repair ; stress fibers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The fluorescent derivative of the actin-binding toxin phallacidin, 7-nitrobenz-2-oxa-1,3 diazole phallacidin, has been used to cytologically demonstrate the presence of actin in lens epithelium, corneal endothelium, and retinal pigment epithelium. In these noninjured tissues, no stress fibers are observed and fluorescence is confined mainly to an area at or near the cell membrane, although some diffuse cytoplasmic staining can also be seen. However, following injury to either the lens epithelium or corneal endothelium of rats and frogs, stress fibers are detected, but only in those cells that migrate into the wound area. Cells on the periphery of each tissue do not partake in would repair and thus maintain their normal appearance. After the tissue has regenerated, stress fibers disappear, and those cells involved in the injury response return to their normal morphology.When rabbit corneal endothelium is placed in tissue culture, stress fibers are observed as the cells migrate away from the initial explant. Upon reaching confluency, these cells spread out and each is surrounded by thick actin-containing bands. Furthermore, they exhibit some stress cables within their cytoplasm. This is in contrast to their appearance in vivo where stress fibers are absent and fluorescence is limited to a region near the cell membrane.
    Additional Material: 4 Ill.
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  • 2
    ISSN: 1432-0878
    Keywords: Capillary ; Endothelium ; Ferritin ; Permeability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.
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  • 3
    ISSN: 1432-0878
    Keywords: Corneal endothelium ; Proliferation ; Actin ; Nitrobenzoxa-diazole, Phallacidin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The distribution of actin in dividing endothelial cells of the rat cornea was studied by fluorescence microscopy by means of the nitrobenzoxadiazole conjugated derivative of the actin-binding toxin phallacidin (NBD-Ph). In normal noninjured tissue, fluorescence is limited to an area at or near the plasma membrane. Twenty-four hours after a corneal freeze injury, stress fibers are detected but only in those cells that are migrating into the wound area. By 48 h post-injury, cells in various stages of mitosis can be identified. During metaphase, anaphase, and telophase, diffuse cytoplasmic staining is observed, although the spindle region remains free of fluorescence. At various sites along the plasma membrane, fluorescence appears stronger compared to other regions. During the latter two stages of proliferation, NBD-Ph positive material can be seen within cell processes. In addition, a band of this material is observed within the region that corresponds to the cleavage furrow. As the daughter cells separate, actin can be detected within the cytoplasmic bridge. The results indicate that NBD-Ph can be used to study the distribution of actin in cells that were proliferating in vivo, and these patterns appear similar to those obtained with immunological methods on cultured cells.
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  • 4
    ISSN: 0730-2312
    Keywords: corneal endothelium ; actin ; tubulin ; upregulation ; autoregulation ; migration ; wound repair ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A single layer of squamous epithelial cells termed the “endothelium” resides upon its natural basement membrane (Descemet's membrane) along the posterior surface of the vertebrate cornea. A well-defined circular freeze injury to the center of the tissue exposes the underlying basement membrane and results in the directed migration of surrounding cells into the wound center. This cellular translocation is characterized by the reorganization of the actin and tubulin cytoskeletons. During migration, circumferential microfilament bundles are replaced by prominent stress fibers while microtubules, observed as delicate lattices in non-injured cells, become organized into distinct web-like patterns. To determine whether this cytoskeletal reorganization requires actin or tubulin synthesis, injured rabbit endothelia were organ cultured for various times and metabolically labeled with 35S-methionine/cystine (250 μCi/ml) for the final 6 h of each experiment. Analysis of actin and tubulin immunoprecipitates indicated no significant increases in 35S incorporation occurred during the course of wound repair when compared to isotope incorporation in noninjured tissues. However, when cytoskeletal reorganization was hampered, either by pre-treating tissues with 7 μM phalloidin to stabilize their circumferential microfilament bundles, or culturing in the presence of 10-8M colchicine to dissociate microtubules, 35S incorporation increased significantly into both actin and tubulin immunoprecipitates at 48 h post-injury. Furthermore, in both cases, exposure to actinomycin D substantially suppressed isotope incorporation. These results indicate that cytoskeletal rearrangement of microfilaments and microtubules during wound repair, in corneal endothelial cells migrating along their natural basement membrane, utilizes existing actin and tubulin subunits for filament reorganization. Disrupting this disassembly/reassembly process prevents cytoskeletal restructuring and leads to the subsequent initiation of actin and tubulin syntheses, as a result of increased transcriptional activity. J. Cell. Biochem. 67:409-421, 1997. © 1997 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 5
    ISSN: 1432-0878
    Keywords: Retinal vessels ; Actin ; Myosin ; Laminin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Actin, myosin, and laminin have been localized in retinal vessels of normal rats by fluorescence microscopy. Actin was localized with the fluorescent F-actin binding toxin nitrobenzoxadiazole phallacidin (NBD-Ph). Indirect immunofluorescence was used to localize myosin and laminin. In addition, laminin localization was also performed with the Protein A-horseradish peroxidase (PA-HRP) method. NBD-Ph staining gave strong fluorescence in both retinal capillaries and larger vessels. Anti-myosin fluorescence could also be observed in trypsin digests of the retinal vasculature. Strong fluorescence of PA-HRP reaction product could be detected in the walls of vessels exposed to antilaminin antibody. Actin distribution in vessels of the RCS rat with inherited retinal degeneration (retinal dystrophic RCS rat) was also studied. After exposure to NBD-Ph, all capillaries showed fluorescence. However, it was more intense in many of the capillaries in the outer retina, which also appeared morphologically abnormal. Electron microscopy of retinal capillaries fixed in 2.5% glutaraldehyde containing 8% tannic acid revealed numerous micro filaments in the pericyte cytoplasm amd some in the basal portion of endothelial cells. In pericytes, these microfilaments are in close association with the endothelial side of the cell. Tangential sections through this region indicate that these filaments may be anchored to the membrane at this site.
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  • 6
    ISSN: 0886-1544
    Keywords: endothelium ; wound repair ; inhibitors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of microfilaments and microtubules during injury-induced cell migration of corneal endothelial cells in situ along their natural basement membrane has been investigated using organ culture. In the noninjured tissue, actin is localized at or near the plasma membrane, whereas tubulin is observed as a delicate lattice pattern throughout the cytoplasm. Twenty-four hours after a circular freeze injury, cells surrounding the wound area extend processes into this region. Fluorescent microscopy using phallotoxins and anti-tubulin antibodies demonstrated the presence of stress fibers and microtubule reorganization within these cells. Between 24 and 48 h post-injury endothelial cells move into the wound region, and by 48 h, the injury zone is repopulated and the monolayer is becoming resstablished. When injured corneas are placed in media containing 5 × 10-7 M cytochalasin B, endothelial cell migration occurs: but it is slow, and wound closure is not complete even by 72 h. In contrast, when tissues are cultured in the presence of 10-8 M colchicine, cell movement is greatly reduced, complete wound closure does not occur, and endothelial cells at the wound edge fail to display extensions typical of migrating cells. Furthermore, when injured endothelia are exposed to 0.05 μg/ml of actinomycin D for 15 min within the first hour after injury and transferred back into culture media lacking the drug for the duration of the experiment, migration does not occur and the wound persists. These actinomycin D treated cells remain viable as shown by their ability to incorporate 3H-uridine and 3H-thymidine. Fluorescence microscopy of actinomycin D treated tissues revealed the presence of stress filaments but disorganized microtubule patterns. Interestingly, 24 h after injury, if the tissue is exposed to actinomycin D, even for periods of up to 1 h, migration is not inhibited. Our results indicate that injury-induced endothelial cell movement appears to be more dependent on microtubule than microfilament reorganization and may require a critical timing of macromolecular synthesis.
    Additional Material: 26 Ill.
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