Neoglycoprotein binding to colorectal tumour cells: Comparison between primary and secondary lesions

Summary Biotinylated neoglycoproteins are useful to determine the expression of sugar receptors (lectins) histochemically in routinely processed tissue sections. Assessment of the presence of distinct receptor classes with specificity to-galactosides and to- or-N-acetylgalactosamine, selected on the basis of their potential relevance for recognition processes within the metastatic cascade in murine model systems, was performed for a common human tumour type, colorectal cancer. The four different types of neoglycoproteins, derived from covalent attachment of commercially available derivatives of-N-acetylgalactosamine, differed only quantitatively in their capacity to detect specific binding on cultured cells and tissue sections, thus posing no major restriction on the choice of synthetic process for histochemical efficiency of the product. Glycocytological application revealed specific probe binding and a regulation of level of receptor expression for a human colon carcinoma cell line primarily forN-acetylgalactosamine-specific receptors upon retinoic acid-induced differentiation. Monitoring of sections of the 12 cases of primary and secondary colorectal lesions invariably disclosed the presence of the respective receptors, the extent of cell labelling in primary tumours and metastases being similar. Establishment of metastases, even in different target organs, is apparently not followed by a major phenotypic variation in this feature.     

Emerging paradigms of T-cell co-stimulation

The analysis of recent data reveals that T-cell co-stimulation is a hierarchical process with elements of mutual interdependence between individual co-stimulators. The expression and function of co-stimulatory molecules is biased on various T-cell subsets and is dependent on the T-cell differentiation state. The classical paradigm of T-cell co-stimulation by professional antigen-presenting cells has to incorporate the newly recognized concept of T-cell co-stimulation in the interaction with peripheral tissues, such as endothelial or epithelial cells. The two signal paradigm of T-cell co-stimulation is being replaced by a multisignal integration concept of central and peripheral co-stimulation.     

Huntington disease-linked locusD4S111 exposed as the α-l-iduronidase gene

-l-Iduronidase (IDUA) has been intensively studied due to its causative role in mucopolysaccharidosis type I (Hurler, Scheie and Hurler/Scheie syndromes). The recent cloning of a human IDUA cDNA has resulted in a reevaluation of the chromosomal location of this gene. Previously assigned to chromosome 22, IDUA now has been localized to 4p16.3, the region of chromosome 4 associated with Huntington's disease (HD). The existence of a battery of cloned DNA, physical map information, and genetic polymorphism data for this region has allowed the rapid fine mapping of IDUA within the terminal cytogenetic band of 4p. IDUA was found to be coincident with D4S111, an anonymous locus displaying a highly informative multiallele DNA polymorphism. This map location, 1.1×10⁶ bp from the telomere, makes IDUA the most distal cloned gene assigned to 4p. However, it falls within a segment of 4p16.3 that has been eliminated from the HD candidate region, excluding a role for IDUA in this disorder.     

15N NMR spectroscopy, 9. Solvent effects on polypeptides and polyamides

Natural abundance ¹⁵N NMR spectra of Nylon-2 to Nylon-8 were measured in 2,2,2-trifluoroethanol, formic acid, trifluoroacetic acid, and fluorosulfonic acid. The ¹⁵N NMR spectra of several sequence polypeptides containing Gly-Gly units were measured in the same solvents depending on their solubility and chemical stability. The shifts of these polymers were compared with each other and strong downfield shifts (up to 20 ppm) were found with increasing acidity of the solvents. The downfield shift was more pronounced in the case of ω-aminoacyl units when compared with α-amino acid residues. ?-Caprolactam shows shift effects that parallel those of Nylon-6. Polysarcosine, poly(L -lysine) ( 5 ), iso-poly(L -lysine) ( 6 ) and the sequence polymer (Tau-?-Aca)_n ( 7 ) were measured in dimethyl sulfoxide, water, formic acid, and trifluoroacetic acid. Polysarcosine, like polyglycine, shows comparatively small shift effects on changing the solvent, and polylysine as well as isopolylysine behave also similarly to other polypeptides, despite their charged side chains. With respect to solvent effects the sulfonamide group of 7 behaves differently from all other amide groups. The solvent effects are mostly explained by hydrogen bonds and protonation of the amide group.     

Serotoninbestimmungen an Thrombocyten und an Fraktionen von Thrombocyten

Zusammenfassung Aus Suspensionen zerstörter Thrombocyten des Menschen wurden reine Fraktionen von Hyalomer und Granulomer hergestellt. Von den Fraktionen wurde jeweils ein Teil für die elektronenmikroskopische Untersuchung, ein anderer Teil für die Bestimmung des Serotonins verwandt. Durch die elektronenmikroskopische Untersuchung jeder einzelnen Fraktion wurde die Reinheit der Fraktionen überprüft. Die Bestimmung des Serotoningehaltes erfolgte am isolierten Rattenmagen. In Kontrollversuchen wurde ferner der Serotoningehalt intakter Thrombocyten bestimmt.Intakte Thrombocyten besitzen im Mittel einen Serotonin-Gehalt von 52 ng/10⁸ Thrombocyten. In den Fraktionierungsversuchen fanden wir 95% des Serotonins in den Hyalomer-Fraktionen und nur 5% des Serotonins in den Granulomer-Fraktionen. In vivo findet sich also der hohe Serotoningehalt fast ausschließlich im Hyalomer der Thrombocyten.

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Summary Pure fractions of hyalomer and granulomer were prepared from suspensions of destroyed human thrombocytes. One portion of the fractions was used for the electron microscopic studies, and the other part for the determination of serotonin (5-hydroxytryptamine). The electron microscopic investigation allowed to check the purity of each single fraction. On the isolated rat stomach the determination of the concentration of serotonin was performed. In control preparations we studied the serotonin concentration of intact thrombocytes. These thrombocytes have a mean serotonin concentration of 52 ng/10⁸ platelets. In fractioning experiments we found 95% of the serotonin in the hyalomer fractions and only 5% of the serotonin in the granulomer fractions. In vivo therefore, the high concentration of the serotonin is found almost exclusively in the hyalomer of the thrombocytes.

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Die Ergebniss wurden vor der Medizinischen Gesellschaft Düsseldorf am 23. 1. 1963 mitgeteilt. Herrn Prof. Dr.Meessen danken wir für die Anregung und Förderung der Arbeit, Herrn Prof. Dr.Greeff für die Hilfe bei den Serotoninbestimmungen.     

Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy

The nuclear envelope functions as a selective barrier separating the nuclear from the cytosolic compartment. Nuclear pore complexes (NPCs) mediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied atomic force microscopy (AFM) to visualize the three dimensional (3D) structure of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another directed against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold-labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antibodies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on NEs. According to the 3D images, the volume of a single gold particle conjugated with secondary antibodies was 10 203 nm³. This volume is equivalent to the volume of 38 IgG molecules associated with one individual gold particle. A similar volume of 11 987 nm³ was calculated from a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identifying antibodies or other macromolecules associated with biomembranes.