The Ets-1 transcription factor is involved in pterygial angiogenesis.

Pterygial pathology is characterized by abnormal corneal epithelial proliferation, stromal modulation, matrix degradation and a strong tendency for otherwise absent corneal vascularization. As the proto-oncogene Ets-1 is known to play a key role in angiogenesis and matrix degradation in other tissues, its involvement in corneal vascularization was investigated. Fifteen pterygia representing two groups were studied. Group 1 consisted of five clinically active pterygia, and group 2 consisted of 10 samples of clinically non-active pterygia. (35)S-labelled ets-1 antisense and sense riboprobes were used for in-situ hybridization of Ets-1 transcription factor in all pterygia. The cytoplasm of blood vessel endothelial cells showed strong expression of ets-1 mRNA in all group 1 pterygia. In contrast, no expression of ets-1 was found in group 2 pterygia. Proto-oncogene ets-1 expression has been shown for the first time in the metaplastic pterygium, an eye tissue of unknown pathogenesis.

Regional glycoprotein expression in the chicken lens.

PURPOSE: Several previous studies have shown that glycoconjugates of extracellular matrix, cell membrane and nucleus play an important role in the mediation of cell proliferation, migration and differentiation. Lens epithelial cells and lens fiber cells show regional differences with regard to these parameters. If glycoconjugates participate in the regulation of these patterns in the lens, there should be regional differences in the expression of glycoconjugates. The investigation was focused on the anterior pole, equator and nuclear bow regions, which differ extensively in lens cell proliferation and differentiation. METHODS: To check this hypothesis, the regional binding pattern of twelve different FITC-conjugated lectins was studied glycohistochemically, using paraffin embedded material. The investigation was focused on the anterior pole, equator and nuclear bow regions. RESULTS: Regional differences in lectin binding patterns were identified in the lens capsule, epithelium and the nuclear bow regions. The lens capsule was fluorescently labeled with GS-I, UEA-I, LPA, MAA, SNA only at the anterior pole and with CON-A, WGA, DBA, SBA only at the equator. Staining of the entire anterior surface of the lens capsule was observed with LFA. Cell membranes of the lens epithelium showed binding of MAA and LFA only at the equator. LFA, LPA, MAA and SNA only stained the nuclei of fiber cells at the nuclear bow region but not of lens epithelial cells. WGA strongly labeled the nuclei of equatorial epithelial cells and fiber cells at the bow region. CONCLUSIONS: It is assumed that the observed regional variations in glycoprotein expression in the extracellular matrix and lens cells contribute to the regulation of cell behavior in different areas of the lens.

Induction of apoptosis and secondary necrosis in rat dorsal root ganglion cell cultures by oxidized low density lipoprotein.

Neural cell degeneration underlies central and peripheral nervous system disorders. In this study we examined the influence of oxidized low density lipoprotein (Ox-LDL) on rat dorsal root ganglion (DRG) cells in culture. Methods used were cell morphology, lactate dehydrogenase (LDH) release, the TUNEL-reaction and DNA fragmentation. Exposure of DRG cells to Ox-LDL for 24 h led to elevation of LDH in the culture medium; short term exposure (4 h) induced apoptosis, evidenced by DNA fragmentation and a positive TUNEL-reaction. DRG cells modified LDL in the presence of Cu2+ to mildly oxidized and to a small extent to fully oxidized forms; these in situ-generated LDL oxidation products were strongly toxic. These results suggest that Ox-LDL is a neurotoxin; it initiates apoptotic cell injury which progresses to necrosis and cell death.

Galactose- binding sites in the acinar cells of the human accessory lacrimal gland.

This investigation for the first time has collected evidence of a specific glycoconjugate contribution of the acinar cells from accessory lacrimal glands to human tears. Amongst group III lectin binding glycoconjugates, monosaccharides seem to be more prominent than disaccharides. alpha (and less obvious beta) Galactose sugar moieties appear to be specifically important. A need for further differentiating investigations is outlined.

Immunocytochemistry of the acellular slime mold Physarum polycephalum. V. Cryosectioning now allows analysis of non-extracted plasmodia of any and all stages.

The spatial distribution of cytoplasmic actin in endoplasmic drops as well as in plasmodial strands can be demonstrated in cryosections by fluorescently labelled phallotoxins and actin antibodies. Our results on cryosections show an identical fibrillar actin distribution as revealed in semithin sections after conventional fixation and embedding. Thus, it is now possible to apply immunocytochemical analysis to any and all plasmodial stages with or without prior fixation and without using extraction procedures. Consequentially the loss of soluble compounds during processing is avoided. The most protective pretreatment of the living specimens before freezing is a 15 min incubation in 1.5 M sucrose containing 50 mM KCl, 10 mM EGTA and 10 mM PIPES buffer, pH 7.0, at 4 degrees C.

Preservation and phallotoxin-staining of the microfilament system in Amoeba proteus.

The spatial organization of the microfilament system as the main component of the cytoskeleton in Amoeba proteus was preserved by a glutaraldehyde-lysine-fixation and visualized with fluorescent phallotoxins (NBD- phallacidin , R-phalloidin). Results obtained by means of this method coincide exactly with observations gained from immunocytochemical, ultrastructural and molecular cytochemical studies, i.e., the microfilament system is mainly displayed beneath the cell membrane, at the hyalo - granuloplasmic border and around the cell nucleus. The preparation procedure employed is suitable for the rapid demonstration of cytoplasmic microfilaments in cells difficult to preserve by chemical fixation.

Cytoplasmic actin patterns in Physarum as revealed by NBD-phallacidin staining.

Fluorescently labeled phallacidin, a F-actin specific drug, was used to demonstrate the morphological variety in the cytoskeletal actin pattern of thin-spread plasmodia of the acellular slime mould Physarum polycephalum. The patterns observed in phallacidin-stained specimens consisted of a polygonal network in the anterior region, and of longitudinal as well as helically twisted fibrils in plasmodial strands of the posterior region. These observations are in complete accordance with our recent results obtained on comparable plasmodia by immunofluorescence microscopy using specific antibodies against actin.

Immunocytochemistry of the acellular slime mould Physarum polycephalum. I. Preparation, morphology, and reliability of results concerning cytoplasmic actomyosin patterns in sandwiched plasmodia.

Small phaneroplasmodia of Physarum polycephalum migrate, under sandwich conditions between two agar sheets and a membrane of cellophane, as thin protoplasmic sheets. This method suitably simulates the situation in the natural habitat of acellular slime moulds; i.e. the narrow clefts of the forest soil. The highly differentiated system of cytoplasmic fibrils displayed under these conditions survives both long-term extraction with glycerol and fixation with methanol, procedures that remove the strong inherent autofluorescence, thus allowing the use of immunocytochemical studies. The complicated fibrillar system of sandwiched plasmodia consists of: (1) a membrane-associated cortical filament layer in the anterior region; (2) a more or less regular polygonal fibrillar network in the intermediate region; and (3) a helically twisted fibrillar system encircling endoplasmic pathways as well as isolated strands in the posterior region. So far, three different cytoskeletal proteins have been identified immunocytochemically as constituents of the fibrillar structures: actin, myosin and AM-protein (fragmin). No positive identification of alpha-actinin, filamin and tropomyosin was obtained using antibodies against vertebrate proteins. Electron microscopy of glycerol-extracted specimens treated with antibodies against actin and myosin revealed that the 6 nm filaments consist of actin, whereas the electron-dense material between single actin filaments appears to be myosin. The AM-protein modulating the polymer status of actin is located in all fibrillar structures.

Pinocytosis and locomotion of amoebae. XIX. Immunocytochemical demonstration of actin and myosin in Amoeba proteus.

The spatial distribution of cytoplasmic actin and myosin in 1. normal locomoting, 2. immobilized, and 3. pinocytosing Amoeba proteus was demonstrated by indirect immunofluorescence microscopy. In orthotactic and polytactic cells fixed during normal locomotion actin is mainly located in a cortical layer delineating the granuloplasm from the peripheral hyaloplasm. In cell areas lacking a hyaloplasmic sheet the actin layer immediately borders the plasma membrane. The amount of actin within the continuous layer seems to increase from the advancing front to the middle cell region and to decrease again toward the uroid. The distribution of myosin is largely congruent to the display of actin, with the exception that the myosin-based fluorescence of the cortical layer gradually increases from the front to the uroid. A considerable amount of actin and myosin is also distributed around the nucleus and the contractile vacuole. In immobilized cells contracted by the external application of 10(-4)M procaine hydrochloride the cortical layer distinctly increases in thickness. In contrast to normal locomoting cells actin and myosin show a uniform distribution within the cell cortex along the entire surface. In pinocytosing cells, up to three cortical layers conspicuously rich in actin are produced during the process of channel formation. One of these layers is located in close proximity to the plasma membrane of the pinocytotic channels and the vacuoles. The immunocytochemical results are discussed with respect to earlier observations on the distribution of actin and myosin in Amoeba proteus as obtained by other methods.

Immunocytochemistry of the acellular slime mold Physarum polycephalum. III. Distribution of myosin and the actin-modulating protein (fragmin) in sandwiched plasmodia.

The acellular slime mold Physarum forms very thin plasmodia when sandwiched between two agar sheets. After extraction with glycerol-containing buffers, suitable objects for immunofluorescence microscopy are obtained, and an analysis of the cytoskeletal and contractile system of Physarum becomes possible. Plasmodia were stained with antibodies against myosin and fragmin, a protein factor involved in actin filament length regulation. The microanatomy and topography of cellular structures containing these proteins were investigated at the light and electron microscopic levels. The patterns obtained with the two antibodies are closely related to those obtained with actin antibody [25]. In both cases the complex system of cytoplasmic fibrils is stained selectively. The fibrils form a more or less regular network in the advancing front zone with the fibrils being interconnected by focal nodes. In the posterior region of the plasmodium, where endoplasmic pathways and protoplasmic veins are differentiated, larger fibrils are detected, running obliquely or longitudinally to the veins. With both antibodies the fluorescent pattern of the fibrils is continuous without indications of periodic interruptions or striations, which would be expected in the case of sarcomere-like subunits. With anti-myosin unstained patches are frequently seen at or close to the nodes of the fibrillar network in the anterior region. The small lobopodia, which are rich in actin, are apparently not stained by the myosin antibody, a result similar to the situation in "ruffling edges¿ of cultured vertebrate cells. Electron microscopic investigations of antibody-labeled fibrils in embedded and sectioned plasmodia allow the identification of antibody molecules at specific sites along the fibrils with a different distribution pattern for each of the two antibodies.

Plasmalemma invaginations of Physarum dependent on the nutritional content of the plasmodial environment.

Quantitative estimates of plasmalemma invaginations in plasmodial veins of Physarum polycephalum were made under different conditions of nutrition. Pronounced differences were observed dependent on the nutritional content of the substrate. There was a decided increase in the number of plasmalemma invaginations in plasmodial veins grown on substrates containing absorbable food substances compared to veins migrating on non-nutrient substrates. This observation supports the proposition that the nutritional content, rather than the physical properties, of the substrate is the decisive factor for the formation of plasmalemma invaginations. The invaginations are believed to be concerned with the uptake of non-particulate food substances.