Pro-invasive gene regulating effect of irradiation and combined temozolomide-radiation treatment on surviving human malignant glioma cells.

The current chemotherapeutic treatment of glioblastoma patients has minor success. Little is known about the molecular and cellular mechanisms of the resistance of gliomas towards current therapies. This study investigated both suppressive cellular effects and regulation of extracellular matrix remodeling proteins with pro-invasive activity in surviving human glioblastoma cells under clinically relevant treatments. All cellular and molecular biological investigations were performed on the genetically well-defined and clinically relevant p53-wild type U87Mg glioma cells. Malignant glioma cells underwent either radiation or temozolomide treatments alone, or combined chemo/radio treatment. Protein expression patterns were investigated by two-dimensional polyacrylamide gel electrophoresis followed by protein spot identification using tandem mass spectrometry analysis. Specific expression levels were quantified by Western-blotting. Extracellular gelatinase activities for both metalloproteinases MMP-2 and MMP-9 were determined by zymogramms. Survival curves indicated no effective suppression of glioma cells under all treatment conditions tested. Morphological changes demonstrated sub-lethal effect of both temozolomide and combined treatment. Expression of MMP-2, MMP-9, and membrane type 1 matrix metalloproteinases (MT1-MMP) was differentially up-regulated by increasing cellular density and treatment conditions. A significantly enhanced extracellular degrading activity under all treatment conditions tested was demonstrated for MMP-2 only. Being a marker for brain tumour progression and angiogenesis, lysozyme c was highly up-regulated under the combined chemo/radio treatment. The activation of proteins with pro-invasive activity indicates an increasing malignancy grade of surviving glioma cells under treatment conditions tested correlating well with more aggressive tumour phenotypes observed clinically in recurrences of treated glioblastomas.

Detection of secretory IgM in tears during rhino-conjunctivitis.

BACKGROUND: With various methods, secretory immunoglobulin M (sIgM) was assessed in tears of patients with rhino-conjunctivitis. METHODS: Tears were analyzed by microimmunoelectrophoresis (MIE), size-exclusion high-pressure liquid chromatography (SE-HPLC), sodium dodecyl sulphate (SDS) electrophoresis and isoelectric focusing. RESULTS: Only very small traces of serum IgM could be found in tears of healthy volunteers. MIE showed that tear sIgM is different from serum IgM. The former migrates in the direction of the anode, while serum IgM migrates to the cathode. The SDS electropherogram of a number of patients showed an additional strong band of the sIgM mu-chain of approximately 68 kDa, after rhino-conjunctivitis. SE-HPLC showed two additional peaks for these patients, at 10.97 min and at 12.94 min, which were attributed to tear sIgM. The former peak consists of a complex of four sIgM molecules. One year later the chromatograms of the former rhino-conjunctivitis patients did not show any peak of sIgM in SE-HPLC. CONCLUSIONS: Serum IgM of a molecular weight (MW) 970 kDa appears only in normal tears at very low concentrations, as a result of transudation from the serum. In contrast, sIgM is synthesized during rhino-conjunctivitis in high concentrations in the human lacrimal glands and the conjunctiva. These increased levels of tear sIgM are due to stimuli for specific protein synthesis.

Decreased gap junctional communication in neurobiotin microinjected lens epithelial cells after taxol treatment.

The aim of the study was to examine gap-junction-mediated intercellular communication after experimentally induced aggregations of microtubules in cultured bovine lens epithelial cells. Intercellular communication between lens cells appears to be crucial for normal lens homeostasis. However, investigations on the maintenance of direct ion and metabolite exchange via gap junctions and its quantified dependency of cytoskeletal microtubules have not been available under conditions leading to bundling of microtubules. Thus, metabolic coupling of neighboring lens epithelial cells was quantified following microinjections of neurobiotin into single cells under various conditions. In controls, intensive gap-junction-mediated intercellular communication could be documented by dye-spreading of microinjected neurobiotin. In contrast, taxol treatment for 1-3 days impaired, but did not completely block gap-junction-mediated intercellular communication. After depletion of taxol, a complete recovery of intercellular communication was achieved. In addition, confocal laser scanning microscopy and rapid-freeze deep-etch electron microscopy revealed a displacement of actin-filaments from the perinuclear cytoplasm, accompanied by an abnormal aggregation of microtubules after taxol treatment, including impeded translocation of connexin 43 from the cytoplasm into the plasma membrane. Incubation of cells with nocodazole destroyed the microtubule network, accompanied by a clear reduction of plasma-membrane-integrated connexin 43 and significant impairment of dye spreading. Thus, in lens epithelial cells intercellular communication at gap junctions made by connexin 43 depends on the integrity of the microtubule network through the translocation of connexins to the plasma membrane.

Redox status of the eye lens: a regional study.

The aim of this work was to study the regional variation of some antioxidant systems in calf lens. Specific lens regions of nearly same age were obtained by a microsectioning technique, and the concentration of reduced and oxidized glutathione, protein sulfhydryl groups, and iron were measured in each lens region. The concentration of reduced glutathione, the major redox buffer in lens, exponentially decreased from the cortical regions to the nucleus. In contrast, the concentration of protein sulfhydryl groups gradually increased from the cortex toward the nucleus. The protein-bound disulfides remained constant throughout the lens. Iron was concentrated in the outer cortical region. The results show that the most dynamic redox-active zone in the lens is the subcapsular cortical region where the oxidant flux meets a highly reducing environment containing a potent redox catalyst.

Tryptophan deficiency arrests chromatin breakdown in secondary lens fibers of rats.

Tryptophan deficiency is known for long time to cause cataract in rats. However, up till now the underlying mechanism is still enigmatic. Histological studies showed an extended lens bow suggesting that the normal breakdown of nuclei in the lens fibres is arrested under these conditions. Using advanced ultrastructural techniques we aimed to clarify this aberrant final differentiation of lens fibres. Albino and pigmented rats were permanently or intermittently raised on a tryptophan deficient diet for 12 and 16 weeks, respectively. Rats of the same age raised on a normal diet served as controls. Lenses were treated for light and electron microscopy. For histology sections were stained for DNA and gamma-crystallins. In addition to routine transmission electron microscopy (TEM), ultrathin sections were subjected to electron tomography and energy dispersive X-ray microanalysis (EDX). Histology verified the extended lens bow for albino and pigmented rats and showed that in the intermittent period of normal diet the fibre nuclei are broken down as in controls. It was further shown that gamma-crystallins are co-localized with DNA in the nuclear domain. TEM revealed that during final differentiation nuclear chromatin becomes highly compacted in a chromosome-like manner and than rapidly evanesces in control rats. This compacted stage persists indefinitely in the tryptophan deficient rats. Electron tomography showed that during differentiation chromatin is first uncoiled to 30 nm solenoids, subsequently to highly compacted 10 nm beads-on-a-string fibrils and than is segregated from the nuclear proteins. EDX revealed that the late stage persisting nuclei consist of domains rich in DNA associated with histones and in domains with mainly proteins. This study corroborates previous findings on the final breakdown of nuclei of lens fibres. It further shows that the chromatin is ultimately uncoiled to beads-on-a-string fibrils and that as the last step chromatin is broken down at this unmasked stage. Except for this last step nuclear breakdown is identical in control and tryptophan deficient rats suggesting that it is not the availability of tryptophan for protein synthesis in general which causes the arrest. Two alternatives for this final arrest are discussed. A low tryptophan content, most pronounced in deeper cortical layers, may inhibit the late synthesis of the DNases and proteases necessary for chromatin breakdown. The radical scavenging by indoleamine 2,3-dioxygenase, which cleaves the pyrrole ring of tryptophan to form formylkynurenine using free oxygen radicals, is impaired by low levels of tryptophan. This decreased scavenging of oxygen radicals will expose the catalytic enzymes for chromatin breakdown, residing in the nucleus in an inactive form for quite a long period, to high levels of oxygen radicals and may affect the activity of these enzymes and therefore the execution of the chromatin breakdown.

Distribution of extracellular matrix proteins in pterygia: an immunohistochemical study.

PURPOSE: This study was carried out to monitor the expression of extracellular matrix proteins (ECMs) and metalloproteinases (MMPs) in pterygial tissue. METHODS: Twenty primary nasal pterygia were studied by indirect routine immunohistochemistry using 13 different primary antibodies against 8 ECMs (five collagens, fibronectin, heparan sulfate, and laminin) fibroblast growth factor (bFGF), von Willebrand factor (vWF), and 3 MMPs (8, 9, and 13). Secondary antibodies were fluoresceinated. Intensity of reaction on individual sections was graded semi-quantitatively. RESULTS: No expression of collagens I, II, and VII was found. Antibodies against collagen III reacted strongly positively (+++) with the entire pterygial stroma. Collagen IV expression was strongly positive in the wall of pterygial blood vessels, moderately positive (++) in the epithelial basement membrane, and only weakly positive (+) all over the stroma. Antibodies against fibronectin reacted moderately positively with stroma, blood vessel walls and epithelial basement membrane. Heparan sulfate was strongly expressed in the blood vessel walls and epithelial basement membrane. Antibodies against bFGF reacted only with pterygial epithelium. Laminin was strongly expressed in blood vessel wall, moderately (++) in the epithelial basement membrane and weakly over the entire stroma. vWF was strongly positive (+++) with pterygial blood vessel walls. Antibody reactions for MMPs differed. It was strong with pterygial epithelium (MMPs 8, 9 and 13), strong to moderate with pterygial stroma (MMPs 8 and 13 versus 9), and absent to weak with pterygial vascular walls (MMPs 8 and 13 versus 9). CONCLUSIONS: This study documents the presence of several ECMs but excludes the expression of others in pterygial tissues. The results especially indicate an active involvement of MMPs 8, 9 and 13 in the pathogenesis of pterygia.

Microinjection of actin antibodies impaired gap junctional intercellular communication in lens epithelial cells in vitro.

PURPOSE: The aim of this study was to check the importance of cytoskeletal actin for gap junction mediated intercellular communication (GJIC) in cultured lens epithelial cells (LEC). METHODS: Bovine LEC were cultured until confluency on cover-slides of a collocate-system. In order to study the cytoskeletal influence on cell communication microcinjection of gap junction permeable neurobiotin into a single cell was preceded by microinjection of actin antibodies. Confocal laser scanning microscopy of specimens treated with actin antibodies and/or subsequent phalloidin labelling, and electron microscopy, were applied to check for cytoskeleton cell membrane links. Specificity of actin antibodies was proved by immoblotting techniques. RESULTS: Immunohistochemistry and phalloidin-rhodamine staining displayed bundles of actin-filaments extending through the entire LEC. Quantitative analysis of GJIC showed intensive dye-spreading of neurobiotin between adjacent LEC. Injection of actin antibodies thirty minutes prior to microinjection of neurobiotin significantly reduced GJIC. Microinjection of irrelevant antibodies had no effect on GJIC. CONCLUSION: Integrity of the actin-cytoskeleton is fundamental for unimpaired GJIC in LEC.

Porcine eye lens crystallins: antigenic similarity with human crystallins and tool for the detection of anti-crystallin antibodies.

BACKGROUND: The usual sources of antigenic material for investigations on circulating immunoglobulins with anti-lens crystallins specificity are saline extracts of human cataract lenses. This practice has a number of drawbacks, especially the possible antigenic alterations that may have occurred in cataract lenses. The aim of this investigation was to compare the antigenic properties of porcine eye lens crystallins and human crystallins, with regard to the possibility for an alternative source of antigenic material for detection of anti-crystallin antibodies in human sera. METHODS: We produced rabbit antisera against saline extracts of human and porcine eye lenses. These sera were applied for the antigenic characterizations of the two extracts with indirect and absorption enzyme-linked immunosorbent assay. The two antigens were further compared by testing them against 30 human sera from cataract patients and 30 sera from blood donors. RESULTS: The antibodies raised against human eye lens cross-reacted with antigens of the porcine lens. This finding was supported by the absorption experiments - the antigens of the porcine eye lens strongly inhibit the reactivity of the rabbit serum raised against human eye lens and vice versa. We established a significant positive correlation (Spearman, r=0.89, P<0.0001) between the reactivity of the tested sera against human and porcine lens extracts. CONCLUSION: These data demonstrated a strong antigenic similarity between human and porcine lens crystallins, suggesting the appropriateness of the use of porcine lens extracts for the detection of humoral anti-lens autoimmune response in patients with eye diseases.

Comparison between modifications of lens proteins resulted from glycation with methylglyoxal, glyoxal, ascorbic acid, and fructose.

Cataract is generally associated with the breakdown of the lens microarchitecture. Age-dependent chemical modifications and cross-linking of proteins are the major pathways for development of lens opacity. The specific alterations in lens proteins caused by glycation with four carbonyl metabolites, fructose, methylglyoxal, glyoxal, and ascorbic acid, were investigated. Decrease in intensity of tryptophan related fluorescence and level of reduced protein sulfhydryl groups, parameters that are indicative for changes in protein conformation, were observed after reaction with all studied carbonyl compounds. Protein carbonyl content, an index for oxidative damage to proteins, was strongly enhanced in methylglyoxal-treated proteins. Cross-linking of glycated proteins was confirmed by polyacrylamide electrophoresis. alpha-Oxoaldehydes were the most reactive in protein aggregation. They also formed specific chromophores absorbing UV light above 300 nm. Significant loss in lactate dehydrogenase activity resulted from incubation with methylglyoxal, followed by glyoxal and ascorbic acid. The results obtained showed that alterations in lens proteins do not follow the specific reactivity of studied carbonyl compounds. Despite the similarity in chemical structures of alpha-oxoaldehydes and ascorbic acid degradation products, they cause specific alterations in lens protein structure with different biological consequences.

Glycated proteins can enhance photooxidative stress in aged and diabetic lenses.

This study intends to clarify the ability of different carbonyl-containing lens metabolites to form advanced glycation end products, which possess photosensitizer activity and to investigate whether these modified proteins could be implicated in lens photodamage. Calf lens protein was experimentally glycated with either methylglyoxal, glyoxal, ascorbic acid, or fructose to obtain models of aged and diabetic cataractous lenses. Being exposed to 200J/cm2 UVA radiation the model glycated proteins produced 2-3-fold more singlet oxygen compared to the unmodified protein and the superoxide radical formation was 30-80% higher than by the native protein. Ascorbylated proteins demonstrated the highest photosensitizer activity. Biological responses of glycation-related photosensitizers were studied on cultured lens epithelial cells irradiated with 40J/cm2 UVA. Tissue culture studies revealed a significant increase in thiobarbituric acid reactive substances in the culture medium of lens epithelial cells after irradiation and treatment with glycated proteins. Lens proteins had a protective effect against UVA induced cytotoxicity, however, this protective effect decreased with the increasing photosensitizer activity of experimentally glycated proteins. The documented glycation-related photosensitization could explain the accelerated pathogenic changes in human lens at advanced age and under diabetic conditions.