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.

Increased DNA breaks and up-regulation of both G(1) and G(2) checkpoint genes p21(WAF1/CIP1) and 14-3-3 sigma in circulating leukocytes of glaucoma patients and vasospastic individuals.

OBJECTIVE: Vascular disorder leading to local ischemia/reperfusion has been shown to play an important role in the glaucomatous damage. A decreased expression level of XPGC-gene has been found in circulating leukocytes of normal-tension glaucoma patients. Although decreased activity of XPGC-gene leads to insufficient DNA-repair, no leukopenia has been observed in glaucoma. Molecular mechanisms ensuring cell survival have not been elucidated yet for glaucoma with vascular disorder. MATERIAL AND METHODS: Using the ex vivo optical imaging method of alkaline "comet assay" comparative quantification of DNA breaks was performed in circulating leukocytes of non-glaucomatous non-vasospastic and vasospastic individuals as well as both normal-tension and high-tension glaucoma patients. Relative expression levels of the anti-apoptotic factors P21(WAF1/CIP1) and 14-3-3 sigma were investigated in all groups tested. RESULTS AND CONCLUSIONS: The quantification of P21(WAF1/CIP1) showed the highest expression rates in high-tension glaucoma patients which were significantly higher than those in all other groups tested. The highest expression rates of 14-3-3 sigma were found in both groups of glaucoma patients. These expression levels correlated well with DNA breaks measured. Since the expression of P21(WAF1/CIP1) in leukocytes was shown to be crucial for their survival under stress conditions, we suppose further that the up-regulation of this gene is the key event in the survival mechanisms of leukocytes in glaucoma accompanied with vascular disorder. The p21(WAF1/CIP1) gene should be further taken into consideration as a potential marker, the up-regulation of which in circulating leukocytes of vasospastic individuals may indicate an increased risk for the developing glaucoma.

Effects of dietary deficiency of selective amino acids on the function of the cornea and lens in rats.

Effects of dietary deficiencies of tryptophan and methionin on the transparency of cornea and lens were investigated in young rats (Brown-Norway, BN; Sprague-Dawley, SD) over 3 months. Transparency of the cornea and lens were evaluated in weekly intervals using a photo-slitlamp microscope. After sacrifice and lens fresh weight determination the lenses were prepared for histopathology. Methionin deficiency had no effect on the parameters investigated. Tryptophan deficiency caused severe loss of body weight in both strains, with additional loss of hair in SD rats. These developed corneal neovascularisations and cataracts. BN rats showed an enhanced zone of discontinuity in the lens. Diet intermission arrested the pathological processes in the eye which restarted when feeding the diet again. This observation is supported by lens fresh weight data. DNA staining evidenced that tryptophan deficiency arrested lens fiber maturation in both strains but stimulated corneal neovascularisation only in SD rats.

Light scattering in normal and cataractous lenses of farmed Atlantic salmon (Salmo salar): a slit lamp and Scheimpflug photographic study.

To investigate normal light scattering and cataract formation, the anterior eye segments of farmed Atlantic salmon (Salmo salar) reared in fresh water and sea water were documented in vivo for the first time with a Topcon SL-45 Scheimpflug camera. A total of 40 fish from the fresh-water-rearing period, obtained from 2 groups of identical age but showing a different growth rate, and 24 fish from the sea-water-rearing period, sampled from 2 groups with identical age but being fed different food brands, were included in this study. The fish were anaesthetized before examination. Due to the naturally wide pupil, no mydriatic compound was applied. All fish were removed from the water for photography, which was performed for each eye in 0 degrees = vertical slit position. Images were recorded on Kodak Tmax 400 black-and-white film. Microdensitometric image analysis of all negatives was performed using a Joyce-Loebl online microdensitometer. In spite of the virtual absence of an anterior chamber gap between cornea and lens and very little light scattering in the normal fish lens, a small number of distinct layers could be reproducibly identified in the lens. While there was little abnormal light scattering which could point to cataract development in young fish from the fresh water period, the evaluation of the lenses from the 2 sea water groups showed the presence of specific forms of cataract especially in the cortical and supranuclear layers. There were significant differences between the groups fed different food brands at the sea water site. In conclusion, Scheimpflug photography proved to be applicable to eye research in fish in vivo. It is suggested that this method should be employed for reproducible documentation as an extension to slit lamp monitoring in experimental research to reveal causative factors for cataracts in farmed fish.

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.

Undergraduate medical education: tendencies and requirements in a rapidly developing Europe.

This study pinpoints the necessity to constantly monitor local approaches in undergraduate medical education on an inter-European scale. Traditional undergraduate medical curricula need restructuring to account for the increasing amount of medical knowledge and rapid changes and developments in societies, nosology, therapy and IT. European undergraduate medical curricula should be harmonized not egalized, with a focus on inter-European sharing of resources, mobility, credit (allocation, accumulation and transfer), definition of European and trans-European mission statements, identification of quality metrics, advice on dealing with conflicting aims such as specialization and generalization, on communicating core knowledge instead of providing overabundance of information, and on introducing multifaceted teaching and learning methods, as well as providing strategies for life long learning. Sound medical education can no longer and nowhere be considered under the autonomous auspices of individual Medical Schools or national philosophies. It has to be perceived and structured as a competitive and flexible approach which promotes life long learning of teachers, students, physicians and other related staff with international awareness. It is stressed that student and staff mobility, as well as virtual mobility in the form of worldwide available teaching modules and expertise have to be incorporated into national medical curricula. This is to guarantee up-to-date education in support of patient demands, future professionality and competitiveness of students, physicians and Public Health System institutions. The formal approaches of traditional subject related curricula as well as problem based learning must be linked with quality approved state of the art ODL, evaluated international CME strategies and training in the utilization of IT in preparation of lifelong learning. Strategies for the use of IT need updating on a regular basis to diminish the gap between undergraduate and postgraduate medical education. General European perspectives of medical education are discussed in relation to ECTS, ODL, compulsory credited and evaluated CME and relicensing of physicians. Prime features of ETM--the most reputed and well-known European medical CME initiative fostering quality assured international awareness are described and recommended for local and nationwide implementation. Specific links of the Bonn undergraduate medical curriculum with credited and evaluated CME and imminent European strategies are detailed. The authors conclude that European universities not adapting at least some of the outlined curricular necessities will rapidly lose their competitiveness compared to other national and international Medical Schools. Harmonized European ethical mission statements and consequent utilization of IT deserve special considerations in this context.

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.

Influence of oxygen free radicals and free radical scavengers on the growth behaviour and oxidative tissue damage of bovine retinal pigment epithelium cells in vitro.

PURPOSE: This investigation was carried out to ascertain whether oxygen free radicals can influence the growth behaviour and consecutive lipid peroxidation of retinal pigment epithelium (RPE) cells in vitro and whether scavengers can counteract these effects. METHODS: The experimental model was based on calf RPE cells. Hypoxanthine/xanthine oxidase (HX/XO) and superoxide dismutase/ catalase (SOD/CAT) served as the radical generating system and scavengers, respectively. The components were tested alone and in combination. Lipid peroxides were determined in culture supernatants by a thiobarbituric acid assay. RESULTS: Concentrations of up to 100 mumol/l of HX alone and 500/ 1000 microU of XO alone, as well as the application of the scavengers without the radical generating system (HX/XO), had no effect. Dose-related reduction of cell growth and increase of lipid peroxidation were found with HX/XO treatment (single dose of 500 and 1000 microU/ml 24 h after seeding). After application of 500 or 1000 microU/ml of XO, CAT, when given alone (1200 U/ ml), counteracted the effect of the radicals on cell growth and lipid peroxidation; SOD (300 U/ml) had no effect. A combination of SOD and CAT was no better than the effect of CAT alone. CONCLUSION: The prevention of radical-induced reduction of cell growth and lipid peroxidation by scavengers supports trials of therapy using antioxidants and/or free radical scavengers for various ocular syndromes with RPE involvement.

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 peroxide level and myeloperoxidase activity in the vitreous of patients suffering from proliferative vitreoretinopathy.

BACKGROUND: Retinal pigment epithelium cells and activated phagocytes are believed to be involved in the pathogenesis of proliferative vitreoretinopathy (PVR). Both cell types are capable of producing oxygen free radicals and other molecules with a high oxidative potential which can lead to a propagation of oxidative damage. It was the aim of this study to investigate whether products of oxidative reactions are detectable in the vitreous body of patients suffering from PVR. METHODS: In vitreous aspirates of patients vitrectomized because of PVR (n = 27), macular pucker (n = 9), or other reasons (controls, n = 31), the following parameters were determined: lipid peroxides (LPO), determined as malondialdehyde-like substances (MDA) and as thiobarbituric acid-reactive substances (TBARS), and myeloperoxidase activity (MPO). RESULTS: Compared with the controls, both LPO levels and MPO activities were significantly elevated in the vitreous of patients suffering from PVR. Vitreous of patients with macular pucker did not reveal any significant differences from controls in the parameters analyzed. CONCLUSION: Our results suggest that both oxygen free radicals and inflammation-related reactions participate in the process of PVR. Oxidative tissue damage is obviously not involved in the pathogenesis of macular pucker.

Growth hormone regulates an N-acetylgalactosamine component in odontogenesis: a specific lectin-binding study in the Lewis dwarf rat.

Dental organs of incisors from normal, dwarf and growth hormone-treated dwarf rats were analysed histochemically using a panel of lectins. A distinctive pattern of differential staining was obtained with Helix pomatia agglutinin, a lectin specific for N-acetylgalactosamine. In Bouin's perfused and paraffin-embedded undecalcified tissues from normal rats, reaction product for N-acetylgalactosamine was visible in the odontogenic cells and some extracellular matrices. In the growth hormone-deficient dwarf rats, the N-acetylgalactosamine reaction was consistently minimal in the odontoblasts, predentin, cementoblasts, cementoid, osteoblasts and osteoid matrices, although the staining of ameloblasts and osteoclasts was similar to normal. Administration of growth hormone to dwarf rats for six days (66 micrograms/100 g rat b.i.d.) restored the reaction for N-acetylgalactosamine in the affected matrices. Thus, an N-acetylgalactosamine rich matric component is differentially expressed during odontogensis. Growth hormone may regulate this component in these matrices, which may be a proteoglycan or a glycoprotein, essential for normal growth of the teeth.

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.

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.

Increased lipid peroxide levels and myeloperoxidase activity in the vitreous of patients suffering from proliferative diabetic retinopathy.

Lipid peroxide (LPO) levels, as determined by high-performance liquid chromatography (HPLC) and by the thiobarbituric acid (TBA) method, and myeloperoxidase (MPO) activity in vitreous of patients vitrectomized because of proliferative diabetic retinopathy were compared with LPO levels and MPO activity in vitreous of patients with no vitreoretinal proliferation. Both LPO levels and MPO activity were significantly elevated in the vitreous of patients with fibrovascular vitreoretinal proliferations secondary to diabetes. The TBA method produced higher values for LPO levels than did the HPLC method. The correlation between the two methods was 0.94. Our results suggest that both oxygen-free radicals and inflammation-related reactions can participate in the pathogenesis of diabetic 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.

Evidence for the prevention of oxidative tissue damage in the inner eye by vitamins E and C.

The injection of oxidative metabolites such as lipid peroxides (LPO) into the vitreous cavity led to tissue damage demonstrable by a dose-dependent increase in intravitreal and intralental LPO levels, which are expressed as thiobarbituric-acid-reactive substances (TBARS). This process was accompanied by an inflammatory response as indicated by the occurrence of leukocyte-derived myeloperoxidase (MPO) in the vitreous body. Intramuscular injections of vitamin E were very effective in reducing both TBARS and MPO activity, whereas vitamin C had a moderate effect on TBARS only. The injection of a combination of the two vitamins was no more effective than treatment with vitamin E alone.