Effects of green tea polyphenols on lens photooxidative stress.
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Animals↗
Biomedical subjects
N S Rafferty
Publications and source records attributed to N S Rafferty.
Lead, learning, and calexcitin in Hermissenda.
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Animals↗
Comparative response to UV irradiation of cytoskeletal elements in rabbit and skate lens epithelial cells.
PURPOSE: This work reports a differential effect of ultraviolet A (UVA) irradiation on the three major cytoskeletal structures, actin and vimentin filaments and microtubules of lens cells in primary culture. The effect on cells from lens of the skate (a bottom-dwelling marine elasmobranch) was compared with that on rabbit lens, in order to assess UVA sensitivity as a function of exposure to these wavelengths in the native habitat. METHODS: Exposure intervals of irradiation time up to 6 hours were selected, at fluences from 13.5 to 54.4 J/cm2 and at 365 +/- 45 nm wavelength, to represent mild to moderate physiological levels. Cultures were fixed and processed with anti-alpha-tubulin-FITC and rhodamine phalloidin, or with anti-vimentin FITC and rhodamine phalloidin conjugates. RESULTS: With epifluorescence microscopy, it was found that microtubules were most sensitive to UVA irradiation (in depolymerizing), followed by actin, with vimentin hardly at all affected. Irradiation for 6 hours followed by incubation for 3 days in fresh medium showed no recovery of actin but good recovery of microtubule organizing centers, followed by mitosis in many (rabbit) cells. Skate cells were more sensitive and showed no recovery. CONCLUSIONS: In view of the role of cytoskeletal elements in intracellular structure, cell division and transport, their disruption supports the hypothesis that UVA may damage lens epithelial cells in vivo so as to contribute to cataract formation. In addition, the data suggest that the lenses of animals exposed to sunlight require effective cytoskeletal repair mechanisms to avoid loss of function.
Actins↗
Agonist-induced rise in intracellular calcium of lens epithelial cells: effects on the actin cytoskeleton.
Primary cultures of rabbit and skate lens epithelia were used to investigate the effect of calcium release from intracellular stores upon the actin cytoskeleton. Primary cultures were loaded with fura-2 AM and intracellular calcium, i.e. (Ca2+)i, quantitated using a Hamamatsu Photonics digital imaging system. Agonists used were bombesin, inositol-1,4,5-trisphosphate (IP3), thapsigargin (Tg), neuropeptide Y (NPY) and calcium chloride. Recordings were typically made on seven cells in each case. We found that IP3 caused a 6-8-fold immediate release of (Ca2+)i in rabbit cells, but skate cells showed no response unless permeabilized with saponin, whereupon an increase of about 50% occurred. Tg induced release from internal stores in rabbit cells, but had no effect on skate cells. Bombesin caused a large increase in (Ca2+)i release in both, while NPY had no effect in either. Skate cells incubated in calcium-free EGTA-Ringer's solution responded rapidly to addition of 5 mM CaCl2, whereas only three of 35 rabbit cells responded, and in gradual fashion. After calcium imaging, the cells were fixed and stained with rhodamine phalloidin or with an antibody against IP3 receptor (IP3R) conjugated to FITC. Fluorescence microscopy revealed that the actin cytoskeleton had reorganized from the normal stress fiber pattern into polygonal networks. Tg caused the same structures to form in rabbit cells, but bombesin had no effect. IP3 receptor was located intracellularly, presumably on endoplasmic reticulum, and was not associated with plasma membranes. The rapid response of rabbit cells may have been caused by the DMSO in which fura-2 was dissolved. We have found an interesting difference in agonist-induced calcium release between rabbit and skate cells. The latter may utilize either a Ca-Na exchanger or capacitative calcium entry, which could reflect a difference in lens accommodative mechanisms. This seems relevant in view of the fact that the rabbit lens accommodates through change in shape, whereas the skate lens does so through translation of position.
Accommodation, Ocular↗
Effects of near UV radiation and antioxidants on the response of dogfish (Mustelus canis) lens to elevated H2O2.
In vitro exposure of dogfish (Mustelus canis) lenses to near-UV energy not incompatible with that of the environment, causes an opalescence that is not present in unirradiated lenses or those irradiated after soaking in alpha-tocopherol or deferoxamine. The ability of whole lenses to destroy H2O2, as shown by their ability to produce O2 bubbles in H2O2 containing media, is markedly diminished by UV exposure without these antioxidant/free radical scavengers added. The lens capsule epithelium is the major site of catalase activity. Bubble formation was prevented by presoaking the lenses in 3-amino-triazole (3-AT), a potent catalase inhibitor. Analytical measurements confirmed the above observations. Near-UV inhibition of catalase may allow H2O2 in the aqueous humor to damage the lens by exerting oxidative stress.
Animals↗
Catalase activity in dogfish (Mustelus canis) ocular tissues.
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Animals↗
Near-UV radiation disrupts filamentous actin in lens epithelial cells.
Ultraviolet radiation in the near range (UVA) causes lens opacification and disrupts the actin cytoskeleton in rabbit and gray squirrel lenses. Changes were noted using transmission electron microscopy of tangential sections and rhodaminephalloidin fluorescence microscopy of epithelial whole mounts of irradiated and unirradiated lenses, and corresponded with gross cataract formation. Irradiated lenses lacked microfilament polygonal arrays at the inner surface of the apical plasma membrane (i.e., in the cell pole next to the lens fibers) in lens epithelia of both species; a condensed actin bundle was present instead. This bundle, and scattered small actin clumps in the cytoplasm, were identified by immunogold TEM, using a specific antibody and a secondary antibody conjugated with colloidal gold. Similar techniques showed breakdown of tubulin and vimentin, but after longer intervals than for the breakdown of actin. Generalized cytologic damage was also present in epithelial cells, but not in the underlying cortical lens fibers. Damage began to occur after 4 hr of irradiation and became more severe with increased exposure. Shielded controls remained clear, had normal cytology and polygonal arrays, and no clumping of actin filaments.
Actin Cytoskeleton↗
The effects of near-UV radiation on elasmobranch lens cytoskeletal actin.
The role of near-UV radiation as a cytoskeletal actin-damaging agent was investigated. Two procedures were used to analyse fresh smooth dogfish (Mustelus canis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringer's medium, and with or without exposure to a near-UV lamp (emission principally at 365 nm; irradiance of 2.5 mW cm-2). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purified polymerized rabbit muscle actin were exposed to the same UV conditions and depolymerization was assayed by ultracentrifugation and high-pressure liquid chromatography. While the two actins studied do differ very slightly in some amino acid sequences, they would react physically nearly identically. The results showed that dogfish lenses developed superficial opacities due to near-UV exposure. Whole mounts of lens epithelium exhibited breakdown of actin filaments in the basal region of the cells within 18 hr of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. SDS-PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized muscle actin in polymerizing buffer led to an increase in actin monomer of approximately 25% in the UV-exposed solutions within 3-18 hr, whether assayed by ultracentrifugation or HPLC. The above indicates that elasmobranch lens epithelial cells contain UV-labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break down the actin structure in these cells. Furthermore, breakdown of purified polymerized muscle actin does occur due to near-UV light exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
Actins↗
Lens cytoskeleton and after-cataract.
Lens epithelial and other ocular cells contain complex arrays of actin filaments which might be expected to allow them to migrate following injury: specifically into the capsular sac in cases of extracapsular cataract extraction or traumatic cataract. To test the possibility, a culture system was developed using a melanotic strain of mice, in which migrating cells are often 'marked' by melanosomes. Injured lenses were cultured on permeable membranes in contact with nutrient medium, and surrounded by iridial tract fragments. After study by light and electron microscopy, it was established that both pigmented and unpigmented cells migrated from the surrounding explants, apparently along the substrate meniscus, to the outside of the lens capsule and then through the wound to the capsule interior. This study suggests a source of cells in development of after-cataract syndrome.
Animals↗
Development of actin polygonal arrays in rabbit lens epithelial cells.
In searching for a clue to the role of actin filament bundles organized into polygonal arrays, or geodomes, in lens epithelial cells, we examined several physical events occurring in the young rabbit lens which may initiate their formation. We used NZW rabbits between the ages of 24 days gestation and 50 days postnatal. Data were obtained from TEM, SEM and fluorescence microscopy. Parameters measured were lens weights, apical surface areas of cells in epithelial whole mounts, epithelial cell thickness, and timing of eyelid opening, breakdown of the tunica vasculosa lentis (TVL) and formation of the ciliary zonules; these findings were correlated with the first signs of development of the arrays. Polygonal arrays formed slowly beginning at one to two days after birth, and with advancing time these thickened and made more numerous connections with the lateral plasma membranes. Development of the arrays was not correlated with onset of vision or disappearance of the TVL or a sudden increase in cell area, since these events occur postnatally at about 9-10 days, nor with the development of zonular fibers since these are already in place at 24 days of gestation. Only lens weights showed a dramatic increase between 24 days gestation and birth. It is surmised that the expanding lens mass may be involved in some way in signaling the organization of actin filaments into geodomes.
Actin Cytoskeleton↗
Immunocytochemical evidence for an actin-myosin system in lens epithelial cells.
Since filamentous actin had been shown earlier to exist in lens epithelial and fiber cells, we inquired whether this could represent a contractile system with myosin and other actin-associated proteins. We resolved this question in freshly removed or organ-cultured rabbit and squirrel lens epithelial whole mounts using immunocytochemical techniques and by immunoblots of extracts separated by electrophoresis. In the former, methods were developed using long fixation times and long incubation in primary antibodies and biotinylated second antibodies visualized by streptavidin immunofluorescence and by diaminobenzidine peroxidase. Myosin was found to be localized along the filamentous rays and at central vertices of polygonal arrays situated at the apices of epithelial cells. It was not clear whether myosin and actin occurred together along the same or adjacent filaments in a bundle. Tubulin and vimentin were found deeper in the cells and were not aligned with actin and myosin filaments. Control lens epithelia treated similarly except for deletion of the primary antibodies showed no staining. As positive controls, pieces of glycerinated sartorius muscle exhibited characteristic cross-banded patterns of actin and myosin when incubated with the same reagents used on the lens epithelium. Denatured extracts of rabbit lens epithelium and of cortical fiber cells separated by electrophoresis and transferred to nitrocellulose paper, stained specifically with the same myosin and tubulin antibodies used in the immunocytochemistry experiments. The molecular weight profile of the myosin polypeptide indicated that lens tissue has myosin II. We conclude that a contractile system exists in lens epithelial and cortical fiber cells, although the function is not understood at this time. We conjecture that the system may act to stabilize lens shape by providing contractile tone.
Actins↗
Comparative study of actin filament patterns in lens epithelial cells. Are these determined by the mechanisms of lens accommodation?
Actin filament patterns in lens epithelia from animals of various taxonomic groups were studied using rhodamine phalloidin fluorescence microscopy of epithelial whole mounts and transmission electron microscopy of tangential sections. The results were compared with the accommodative mechanism operating in each case as reported in the literature. Lenses that accommodate by deformation of the anterior surface, in squirrel, chipmunk, rabbit, monkey and human, showed polygonal arrays (PAs) at the apical end of the epithelial cells. Lenses that translate as a whole, in shark, bony fish and frog, showed stress fibers (SFs) at the basal or apical end of the cells. No specialized actin pattern was seen in turtle and bird, which have lenses that are squeezed into an anterior lenticonus; cat, where the lens is translated forward; or rat, cow and most mice, which have no defined accommodation. In exception, certain strains of laboratory mice did show sequestered actin bundles (SABs) and/or PAs. Based on our findings, we conclude that PAs, which resemble geodesic domes, do not take an active part in near-point accommodation; but like SFs, may serve to resist overextension by internal pressure of the fiber mass or by zonular tension.
Accommodation, Ocular↗
Actin filament patterns in mouse lens epithelium: a study of the effects of aging, injury, and genetics.
Using mainly fluorescence microscopy after rhodamine-phalloidin staining, the F-actin distribution in the mouse lens epithelium was studied with regard to the effects of age, genetic strain, and mechanical injury. These studies have revealed that aside from its association with the plasma membrane the structural organization of F-actin in the mouse lens epithelium in situ is characterized by two major configurations: (1) a filamentous arrangement in such patterns as stress fibers, polygonal arrays (PAs), and meshworks, and (2) a highly concentrated structure called a sequestered actin bundle (SAB). The aging study indicated that the SAB is a consistent character in C57BL/6 mice from the age of 5 wk on, but not in CF1 mice. The size and shape of the SAB change gradually with age as inferred from two-dimensional measurements. The genetic study on the SAB character using hybrids and congenic strains showed that it is inherited as a Mendelian dominant, probably multigenic mode. Finally, the injury study revealed a structural modification in cells around the wound, including flattening of cells at the edge and extension of processes into the wound space. In the rest of the epithelium, injury amplified membrane infolding and fluorescence of polygonal arrays but diminished the size and fluorescence intensity of SABs. These changes are thought to be correlated with wound repair involving cell division and migration. These studies illustrate the variability in F-actin expression in situ in lens epithelial cells that can be induced by intrinsic and extrinsic factors.
Actin Cytoskeleton↗
Immunogold-EM localization of actin and vimentin filaments in relation to polygonal arrays in lens epithelium in situ.
An indirect immunogold technique for transmission electron microscopy was used for localizing two cytoskeletal proteins, actin and vimentin, in the epithelium of freshly removed rabbit lens, especially in relation to the polygonal array structures located at the apices of the epithelial cells. Antibody specificity was determined on semi-pure chicken breast muscle actin and bovine lens vimentin using Western blotting of these proteins and extracts of rabbit lens epithelium separated by SDS-PAGE. Whole lenses of rabbits were lightly fixed in glutaraldehyde and embedded in LR White resin. Tangential sections were taken at 70 to 80 nm and at 0.25 micron and used for single-labeling, and double-labeling with antibodies raised in different hosts and treated with appropriate second antibodies conjugated with non-overlapping sizes of gold particles. Routine and stereomicroscopy were used to analyze gold-label patterns. The study shows that the rays of the polygons project deeply into the cell from the vertices lying on the inner apical membrane. Actin is located on the filaments of rays, but vimentin is not associated with the polygons at the level in the cell that we studied. Vimentin filaments are found in deeper regions of the epithelial cell. Stereopairs were useful in differentiating where the gold-label was located and in fact, this technique demonstrated that most of the label is on the surface of sections where the filaments are exposed.
Actins↗
Light conductance in the ocular lens.
The collimated beam of a He-Ne laser aimed tangentially at the equator of submerged bovine or rabbit ocular lenses enters the lens and travels in the surface of the lens. We suggest that the effect is the result of light conductance along the interface between the bulk of the lens (including the lens epithelium on the anterior surface) as the medium of higher refractive index and the capsule as a medium of lower refractive index. Light conductance can be demonstrated in isolated lenses as well as in intact eyes. It is very sensitive to alterations of the state of the lens and therefore it may offer a new method to study very early stages of lens damage and cataract formation.
Animals↗
Polygonal arrays of actin filaments in human lens epithelial cells. An aging study.
In order to determine the importance of lens actin filament configuration to lens accommodation, the pattern of actin filaments in the epithelium was studied in human lenses from different decades of life spanning the accommodative and non-accommodative years. Polygonal arrays of microfilaments were demonstrated in whole mounts of epithelium from normal and cataractous lenses using rhodamine phalloidin, an actin-specific, fluorescent-labeled probe. Tangential section transmission electron microscopy (TEM) studies confirmed that these arrays consist of central vertices and interconnecting filament rays, which line the apical end of each epithelial cell and appear to attach to the lateral membrane. These polygonal arrays were present in human lenses ranging from 25-94 yr of age. Measurements of intervertex distance showed remarkable constancy throughout the ages studied. In view of these findings, it is proposed that a possible function of these polygonal arrays is to stabilize the lens epithelium during lens flattening.
Actins↗
Actin in polygonal arrays of microfilaments and sequestered actin bundles (SABs) in lens epithelial cells of rabbits and mice.
The pattern of localization of actin filaments was compared in whole mounts of lens epithelium of rabbit and mouse using the fluorescently-labeled actin-specific probe, rhodamine-phalloidin. In the adult rabbit lens, fluorescent polygonal arrays consisting of central vertices and interconnecting filaments were present in the apical end of each epithelial cell. Electron microscopy confirmed that these arrays lined the cytoplasmic side of the apical membrane. In the mature adult mouse, polygonal arrays were not seen either with fluorescence or electron microscopy. Instead, the actin was packaged in a single, elongated, often curved bundle near the epithelial cell apex, referred to as a "sequestered actin bundle" or SAB. The SAB often appeared attached to the plasma membrane and to approach the perinuclear basket of microfilaments. The significance of the differences in these two patterns of actin is discussed in terms of differences in the accommodative ability and static lens shape in these two animals.
Accommodation, Ocular↗
Lens wound healing and cataractogenesis in a pigmented eye.
Light and electron microscopy and slitlamp microscopy were used to follow the development and partial repair of injury-induced cataract in the lens of the pigmented eye of the grey squirrel. These processes proceed in much the same way as in previously-studied albino rodents and rabbits in spite of invasion of the lens opacity by pigmented cells in the squirrel eye. Epithelial and capsular regeneration and lens fiber repair occur rapidly and apparently independently from the accumulation of pigmented cells, fibroblasts and collagen in the wound outside the lens epithelial layer. Using morphological criteria, some of the pigmented cells in the lens wound are identified as iris stromal melanocytes and pigmented epithelial cells; this is consonant with the slitlamp observations of streams of pigmented cells extending from the iris to the lens wound. The role, if any, of the pigmented cells in lens wound healing is unknown.
Animals↗