Uwe Pfüller

The cytotoxic effect of mistletoe lectins I, II and III on sensitive and multidrug resistant human colon cancer cell lines in vitro

Multidrug resistance glycoprotein1 (MDR-1) eliminates amphiphilic chemotherapeutic agents out of tumour cells leading to therapeutic failures. The aim of this study was to investigate the cytotoxic effect of mistletoe lectins (MLs) I, II and III on the sensitive human colon cancer cell line HT 29(mdr-), its multidrug resistant variant HT 29(mdr+), the variant HT 29(SF1m) transfected with the MDR-1 gene and its sensitive control cell line HT 29(deltaSF). Both cell proliferation and ML binding pattern were analysed. Marked quantitative differences concerning the cytotoxic effect of the three MLs on the different cell lines were observed. All MLs showed the greatest cytotoxicity towards the HT 29(mdr+) cells, in which multidrug resistance (MDR) was induced by increasing concentrations of a MDR inducing agent. In contrast, MDR-1 and mock-transfected cells showed almost the same sensitivity towards the three MLs as the control cells (HT 29(mdr-)). FACS analysis showed that the HT 29(mdr+) cells were the cells with the highest density of ML binding sites. Thus, higher sensitivity of HT 29(mdr+) cells are not caused by the overexpression of MDR-1, but are caused by the general changes of the cellular glycosylation during the acquisition of the MDR phenotype.

Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I

Abstract The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay. The results indicated that ML-I has a broad range of affinity for Gal, α, β linked sequences. The galabiose (C, Gal α 1→4Gal) sequence, a receptor of the uropathogenic E. coli ligand, was one of the best disaccharide inhibitors tested. The lectin also exhibits affinity for Lac(Gal β 1→4Glc), T(Gal β 1→3GalNAc), I / II (Gal β 1→3/4GlcNAc) and B(Gal α 1→3Gal) sequences. Gal α 1→4Gal and Gal β 1→4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment. This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.

Biochemical, histochemical and cell biological investigations on the actions of mistletoe lectins I, II and III with human breast cancer cell lines

Cytotoxicity of the mistletoe lectins I, II and III towards six human breast cancer cell lines was assessed using the Mossman assay. In addition, binding of the three mistletoe lectins to the separated membrane glycoproteins of these cell lines, the binding and uptake of these lectins into the cells in tissue culture and the binding of the lectins to histological preparations of these cell lines were analysed. The results indicate that there are quantitative differences concerning the toxicity of these three lectins towards the different cell lines. Furthermore, the lectin binding pattern in the cell lines differed. In Western blots, several membrane glycoproteins were labelled by the lectins. Our results indicate subtle differences between the three lectins with regard to the parameters mentioned above; however, the toxicity of all three lectins from mistletoe is so similar that they all seem suitable for the construction of immunotoxins.

Binding patterns of mistletoe lectins I, II and III to microglia and Alzheimer plaque glycoproteins in human brains

Glycoconjugates of microglial cells and in some cases those glycoconjugates present in the amyloid plaques in Alzheimers disease in the cerebral cortex can be stained with a lectin from mistletoe (ML-I) using a labour-intensive and time-consuming indirect immunoperoxidase technique. In order to simplify the staining method and to test the staining characteristics of the other recently isolated mistletoe lectins (ML-II, ML-III) biotinylated MLs I-III were used together with an avidin-alkaline phosphatase-complex for visualisation. Our findings indicate that this new improved technique can also be used for detection of microglial cells and is considerably faster than the old method. In addition to microglial cells, ML-I labelled plaque glycoproteins possibly indicating that glycoconjugates derived from microglia can be detected in plaques. In contrast to ML-I, both ML-II and ML-III did not stain microglial cells.

Distribution of negative charges of rat brain synaptosomes established by means of protamine-ferritin conjugate

The negative electric charges on the synaptosomes from rat cerebral cortex were studied by means of protamine-ferritin conjugate. The synaptic vesicles in some synaptosomes were heavily labelled with the positively charged conjugate. The synaptosomal membranes including presynaptic and postsynaptic membranes were also stained but in a lesser degree. It was established that the major dense line and the intraperiod line of myelin contaminants were labelled, too.

Elektronenmikroskopische Darstellung saurer Mukopolysaccharide mit ferrocenhaltigen Kationen

Zusammenfassung Ferrocenhaltige Kationen vom Invertseifentyp werden von sauren Mukopolysacchariden selektiv gebunden. Der Nachweis der Substratfixierung kann lichtoptisch (Sudanophilie) und elektronenmikroskopisch (eisenbedingter Kontrast) erfolgen. Von den insgesamt 39 synthetisierten ferrocenhaltigen Invertseifen ergaben mindestens 5 eine gute Kontrastierung saurer Mukopolysaccharide. Der starkste Kontrastierungseffekt wurde fur Cetyl-dimethyl-ferrocenylmethyl-am-moniumchlorid gefunden.

Visualization of anionic sites using polyethyleneimine-metal complexes

17 Polyethyleneimine-metal complexes were synthesized and 3 of them were tested cytochemically for visualization of negative tissue charges. The demonstration of the anionic sites was carried out on rat cerebral cortex and on frog cutaneous pectoral muscle. As controls, neuraminidase digestion, methylation, and omission of osmium-postfixation were used. The osmiophilic properties of polyethyleneimine, polyethyleneimine salts, and polyethyleneimine-metal complexes were discussed.