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.

Non-sufficient cell cycle control as possible clue for the resistance of human malignant glioma cells to clinically relevant treatment conditions.

OBJECTIVES: Human gliomas have a catastrophic prognosis with a median survival in the range of one year even after therapeutic treatment. Relatively high resistance towards apoptotic stimuli is the characteristic feature of malignant gliomas. Since cell cycle control has been shown to be the key mechanism controlling both apoptosis and proliferation, this study focuses on DNA damage analysis and protein expression patterns of essential cell cycle regulators P53 and P21waf1/cip1 in glioma under clinically relevant therapeutic conditions. MATERIAL AND METHODS: U87MG cell line, characterised by wild p53-phenotype relevant for the majority of primary malignant glioblastomas, was used. Glioma cells underwent either irradiation or temozolomide treatment alone, or combined radio/chemo treatment. DNA damage was analysed by the "Comet Assay". Expression rates of target proteins were analysed using "Western-Blot" technique. RESULTS AND CONCLUSIONS: "Comet Assay" demonstrated extensive DNA damage caused by temozolomide treatment alone and in combination with irradiation, correlating well with the low survival rate observed under these treatment conditions. In contrast, irradiation alone resulted in a relatively low DNA damage, correlating well with a high survival rate and indicating a poor therapeutic efficiency of irradiation alone. Unusually low up-regulation of P53 and P21waf1/cip1 expression patterns was produced by the hereby tested stressful conditions. A deficit in cell cycle control might be the clue to the high resistance of malignant glioma cells to established therapeutic approaches.

Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro.

PURPOSE: To compare effects of calpain inhibitors on in vitro light-scattering in rat lens soluble protein and calcium-ionophore (A23187)-induced cataract formation in cultured rat lenses. METHODS: Rat lens soluble protein was hydrolyzed for 24 hours by activation of endogenous lens calpain. Ten calpain inhibitors were tested in this model at 10 and 25 microM concentration. As an index of protein precipitation, light scattering was measured daily at 405 nm for 8 days. Lens proteins were analyzed by isoelectric-focussing. Subsequently, rat lenses were cultured for 5 days with 10 microM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 microM concentration in this model. Cataract evaluation, isoelectric-focussing and calcium determinations were performed. RESULTS: At 25 microM concentration AK295, SJA6017, E-64, PD-150606 and MDL28170 produced greater than 25% inhibition of light-scattering. Isoelectric-focussing revealed that addition of Ca(2+) produced characteristic crystallin proteolysis and aggregation patterns. AK295, SJA6017, MDL28170 and E64c prevented these changes. Lenses cultured in A23187 exhibited nuclear cataract, elevated calcium and proteolysis and aggregation of crystallins. Co-culture with SJA6017, MDL28170 and E64c reduced A23187-induced nuclear opacities, proteolysis and aggregation of crystallins without affecting increased total calcium. CONCLUSIONS: Endogenous calpain-activation model and A23187-induced cataract model can be used sequentially to screen calpain inhibitors for potential anti-cataract activity. Proteolytic changes in lens cortex after exposure to A23187 are also due to calpain activation. AK295, SJA6017 and MDL28170 possess efficacy against calcium-induced models of rodent cataracts. Use of calpain inhibitors represents a promising approach to cataract therapy.

Expression of the neural cell adhesion molecule in mouse taste buds after denervation.

Expression of the neural cell adhesion molecule (NCAM) was studied by use of an immunocytochemical technique in the taste buds of mouse circumvallate papillae after bilateral transection of the glossopharyngeal nerves. In untreated mice, innervated type-III cells reacted with anti-NCAM antibody. After denervation the taste buds gradually decreased in number and size, and were practically absent within 11 days. In parallel, NCAM-reactive cells decreased at 3 and 8 days after surgery and at 11 days they were no longer found. Three days after denervation, synaptic contacts between type-III cells and nerve fibres were not found because of the disappearance of nerve fibres. However, remaining type-III cells, characterized with dense-cored vesicles, still maintained NCAM expression on their plasma membrane until day 8.

Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C.

BACKGROUND: Isoelectric focusing (IEF) of tear proteins has not yet been carried out in a satisfactory way. Two-dimensional (2D) electrophoresis, especially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tear proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. METHODS: Ampholines, covering a broad range from pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubation with a group II lectin and two group V lectins was performed. RESULTS: Tear proteins could be separated into 31 single bands. Tear-specific pre-albumin (TSPA), lactoferrin, sIgA, IgG and lysozyme were found to be main components. Isoelectric points (IEPs, pls) of all proteins separated were determined by comparison with IEF standards. 2D patterns of IEF and SDS electrophoresis were obtained for the main subunit components of lactoferrin, sIgA, TSPA, and lysozyme. An additional new component of considerable concentration was focused at pI 8.6 with a subunit MW of 14 kDa. With s-WGA a component at an IEP of 5.2 was visualized, representing transferrin. With SNA, lactoferrin stained as a sharp main band at pI 5.1 with three additional weaker bands at IEPs from 4.8 to 4.9. At IEPs between 4.4 and 6.1, multiple components of sIgA were stained with MAA. The sugar specificity of transferrin at pI 5.2 was beta-GlcNAc. Lactoferrin showed glycation with NANA-alpha-2-6-Gal or NANA-alpha-2-6-GalNAc, whereas the sugar specificity of sIgA was NANA-alpha-2-3-Gal. CONCLUSIONS: The investigative strategy applied here, including IEF alone, in combination with SDS-electrophoresis, and SDS-electrophoresis followed by lectin staining proved to be a reproducible method for tear protein analysis of hitherto unexperienced capacity. Lectin-stained bands of native tear proteins are not uniformly glycated by one sugar residue, but show various sugar specificities. IgA as a whole molecule is specifically glycated with NANA-alpha-2-3-Gal.

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.

Effects of UV-B on the growth pattern of bovine passage I and II lens epithelial cells in vitro.

To further evaluate the pivotal role of epithelial cell proliferation in lens homeostasis, this study compares the principles of regional growth control in central and preequatorial bovine lens epithelial cells in culture. Central lens epithelial cells do not proliferate in vivo while preequatorial cells do. In tissue culture, both central and preequatorial cells proliferate, albeit at different rates. At all stages investigated, nonirradiated passage I central cells proliferated less extensively when compared to the preequatorial cells. In contrast, central passage II cells showed a higher proliferation rate than peripheral cells until day 7, when the situation reversed. UV-B radiation led to a dose-dependent reduction of cell proliferation but did not change the principle cytokinetic differences between central and peripheral cells in passage I cells. Under all circumstances, passage I cells grew more intensively than their passage II counterparts. Data on origin-related differences in cell proliferation of cultured lens epithelial cells suggests growth control features other than just the regionally limited expression of growth factor receptors in the preequatorial extracellular matrix and cell membranes. Investigations are seen as an important step towards a better understanding of the features underlying regional proliferation control and its impairments, at least in lens epithelial cells.

Increased lipid peroxides and inflammatory parameters in the retina adjacent to choroidal melanoma.

Oxidative tissue damage and inflammatory reaction were investigated in the neurosensory retina of five eyes with malignant choroidal melanoma and correlated to the distance from the tumour. The results showed elevated levels of both lipid peroxides (expressed as thiobarbituric acid reactive substances) and myeloperoxidase in the retina adjacent to the tumour. The values declined with distance from the tumour. The results indicate that production of oxygen free radicals contributes to tumour associated retinopathy.

Developmental changes in the distribution of cecal lectin-binding sites of Balb-c mice.

The existence of lectin-binding sites was investigated in the cecum of Balb-c mice at seven developmental stages ranging from 18 days post conception (p.c.) to 8 weeks after birth. Nine horseradish-peroxidase-conjugated lectins (concanavalin A, Triticum vulgaris, Dolichus biflorus, Helix pomatia, Arachis hypogaea, Glycine maximus, Lotus tetragonolobus, Ulex europaeus, Limulus polyphemus) were applied to 5- to 7-microns thin paraffin sections of Bouin-fixed tissue. After DAB staining the sections were evaluated by light microscopy. It was shown that each lectin exhibits a unique developmental pattern. The adult binding patterns were established at the age of 3-4 weeks with only minor changes occurring thereafter. Considerable differences in binding patterns occurred not only between lectins of different groups but also between lectins with the same nominal monosaccharide specificity.

Morphological investigations on the small intestinal mucosa of mouse after mild hyperthermic treatment.

Local hyperthermia, when applied as a therapeutic agent against radio-resistant tumours, can result in serious side effects in adjacent non-tumorous tissue, one especially thermo-sensitive organ being the small intestine. An experimental morphological study was therefore undertaken with mice to investigate whether "mild hyperthermia" (41 degrees C for 15, 30 and 60 min) causes alterations at the mucosa of the small intestine, and if so what are these effects and their likely pathomechanisms. Descriptive light and electron microscopical studies and morphometric evaluations are reported on the jejunal mucosa. The observed changes were most extensive after 60 min of 41 degrees C hyperthermia; but even 30 and 15 min hyperthermic treatment was followed by severe degenerative changes. The epithelium of the basal crypts and the stromal cells seemed to remain morphologically unaffected under these conditions, though the stroma can react with a temporary contraction. No "prime event" in the hyperthermia provoked tissue changes can be deducted from the combined light, electron microscopical, and morphometric studies. Comparing, however, the extent of the effects after mild hyperthermia of 60, 30 and 15 min, respectively, four phases of intestinal alterations due to mild hyperthermia and a likely pathomechanism of this treatment can be defined. The clinical implications of the findings are discussed.

Growth hormone receptor expression in the Dunning R 3327 prostatic carcinoma of the rat.

The Dunning R3327 rat carcinoma is an important model for human prostate adenocarcinoma. In the present study this tumor was further characterized by immunohistochemical demonstration of receptors for growth hormone (GH-R). Weak GH-R immunoreactivity was present in the secretory epithelial cells of the tumor acini. Large epithelial cells which were localized at the periphery of the acini and large cells in the stroma, which are probably derived from the epithelium ("Large neoplastic epithelial cells"), displayed a strong staining with one of the monoclonal antibodies (Mab 263) to GH-R. The presence of GH-R receptors in proliferating prostatic tumor cells supports the concept that GH reacts directly on prostate target tissue to facilitate tumor cell growth.

Freeze-fracture analysis of the respiratory cilia from the bronchial mucosa of a patient with primary ciliary dyskinesia.

Respiratory cilia of the bronchial mucosa from a 5-year-old boy with clinical evidence of classical Kartagener's syndrome (situs inversus, bronchiectasis and sinusitis) were first examined by means of transmission electron microscopy for identification of the axonemal defects described as typical for primary ciliary dyskinesia (PCD). Additional oscillography was performed on the cilia in vitro, which showed absence of a coordinated ciliary beat frequency. After clear classification of the case as PCD, a freeze-fracture examination of the respiratory cilia was performed, which revealed a higher density of intramembrane particles on the outer fracture face (E-face) than on the inner fracture face (P-face). The results were discussed with regard to probable pathogenetic aspects on PCD.

Differential immunocytological expression of a paraprotein-associated idiotype is possibly related to variant immunoglobulin molecules in plasmacytoma cells.

An anti-idiotype antibody (anti-Id Ab) was made by immunization of a rabbit with the IgG-2/lambda paraprotein from a plasmacytoma patient (Sa). The antibody was directed against determinants of heavy (H) and light (L) chains on the F(ab)2 piece of the paraprotein. Using this antibody an unusual pattern of cytoplasmic fluorescence was seen with bone marrow cells of the patient. One population (type I) showed reactivity in association with gamma and lambda chains, the other (type II) with the anti-Id Ab only. So, by immunological methods, biclonality could be demonstrated using the anti-Id Ab but not with anti-isotype antibodies. L chains of mol. wt 23.5 and 17.0 kd were observed. The H and L chains of small molecular weight were not detected in the patient's serum nor in the culture supernatant. Thus, deleted and intracellularly degraded H and L chains may cause the unusual staining pattern of the type II cells.

Cell migration from the chick olfactory placode: a light and electron microscopic study.

The differentiation of the olfactory placode in the chick has been studied using light and electron microscopy. Special attention was paid to the appearance of neuronal cells within the placodal ectodermal thickening, the migration of cells out of this tissue and the appearance of the first fila olfactoria in the differentiating olfactory mucosa. Between the third and fifth day of incubation a large number of cells is observed leaving the base of the invaginating olfactory placode, often in contact with thin axon bundles. These cells are characterized by a well-developed Golgi apparatus, a considerable number of mitochondria and dense-core vesicles. The morphology of these migrating cells resembles that of cells observed near the basement membrane within the developing olfactory epithelium and is clearly different from the mesenchymal cells which are filled with polyribosomes. At the sixth day of incubation thick axon bundles can be observed within the epithelium and the underlying lamina propria. The possible fate of the migrated epitheloid cells is discussed.

Spatial pattern of sensory cell terminals in the olfactory sac of the tiger salamander. I. A scanning electron microscope study.

The olfactory sac of adult tiger salamanders (Ambystoma tigrinum) was assessed by scanning electron microscopy and the hitherto unknown variety of the olfactory sac cells has been documented. Regional differences in respect to the composition of the apical epithelial surface border in the olfactory sac have been recorded. Transitions between the olfactory epithelium proper and epithelia with different surface structures have been described. It has been shown that both the roof and the floor of the olfactory sac are mainly covered by sensory epithelium with a surface of uniform appearance. However, the sensory area of the floor is interspaced with non-olfactory epithelial bands. Variations were also noted in regard to secretory cells, cells with conical profiles, microplicae arrangements and populations of microvilli and kinocilia. These differences have been discussed in relation to electrophysiological data which suggest a regional patterning in the response to odours.

Effect of N-methyl-formimino-methylester on the vomeronasal neuroepithelium of mice.

N-methyl-formimino-methylester (MFM), a highly volatile chemical substance, causes massive, transient sensory-cell degeneration in the main, but not in the vomeronasal olfactory sensory epithelium of mice. After MFM-treatment it appears possible to study the accessory olfactory system after chemical "deafferentation" of the main system.

[Epithelial surface of trachea in adult male albino rats (author's transl)]. / Die Oberflächenmorphologie des Trachealepithels männlicher Albinoratten. Eine reflektionsrasterelektronenmikroskopische Studie

The epithelial surface of trachea in adult male albino rats is studied by reflection scanning electron microscopy (REM). Surface morphology of the tracheal epithelium shows extensive individual and regional variability. Identical surface morphology may result by different causal factors. Regional morphology is presented by a mapping of the epithelial surface. Special attention is given to the structure of the cilia. Furthermore the morphology and distribution of possible receptor cell terminals is shown. The validity of reflection scanning electron microscopic studies for identifying the patho-histogenetic action of air pollutants is discussed.