Hans Georg Fromme

Metabolism of sulfated glycosaminoglycans in cultured endothelial cells and smooth muscle cells from bovine aorta.

The glycosaminoglycan metabolism of cultured endothelial cells and of cells grown from the intima and from the media layer of bovine aorta thoracia was investigated in a comparative study. The following results were obtained: 1. Endothelial cells have in common with intima and media cells the distribution of newly formed sulfated glycosaminoglycans into extracellular, pericellular and intracellular compartments. Endothelial cells, however, synthesize lower amounts of glycosaminoglycans and distribute them in a different ratio into the three pools. 2. Though all the various cell lines synthesize chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and small amounts of keratan sulfate, endothelial cells exhibit a unique distribution pattern of sulfated glycosaminoglycans in each of the three compartments. Generally, a high proportion of heparan sulfate and chondroitin 6-sulfate and a very low dermatan sulfate content was detected. 3. Heparan sulfate produced by endothelial cells has a higher N-sulfonate content when compared with that from other sources. The cell membrane-associated heparan sulfate, especially, exhibits some heparin-like features as judged by nitrous acid degradation and susceptibility towards heparitinase.

Localization of sulfated glycosaminoglycans within cell nuclei by high-resolution autoradiography.

Abstract Skin fibroblasts cultured from patients with Sanfilippos disease type B and Hurlers disease, preincubated with [35S]sulfate, were subjected to high resolution autoradiography. A 4-day pulse followed by a 24 h chase resulted in a labelling of 61% of the cell nuclei by silver grains, thus indicating the existence of ethanol-insoluble [35S]sulfate-containing material within the nuclei, especially within their chromatin-rich peripheral zone. A computer-calculated statistical evaluation of the autoradiographic results showed that the silver grains overlying the cell nuclei originated from [35S]radioactivity within the nuclei and not from an overall background or cross fire effects of cytoplasmic radiation sources. Chemical analyses of chloroform/methanol extracts and pronase digests of the [35S]labelled cells provided evidence that neither [35S]sulfatides nor [35S]glycopeptides contribute substantially to the cellular [35S]radioactivity. The results strongly suggest the association of sulfated glycosaminoglycans with cell nuclei.

Immunoelectron-microscopic study on the location of fibronectin in human fibroblast cultures

By immunoelectron-microscopic techniques and the recently developed carbon-film cytochemistry it was possible to localize fibronectin or fibronectin-containing material on cultured human skin fibroblasts. In carbon-film replicas of cell surfaces this material is arranged in the form of a fibrous network. In ultrathin sections, this network appears as rather bulky patches on the cell surfaces. In addition fibronectin is often observed in the interspaces between neighboring cells. This finding supports the assumption that fibronectin is involved in the mechanisms of cell-cell contact, cell-substrate adhesion, and cell recognition.

Electron microscopic localization of concanavalin A receptor sites in pollen surface material after fixation with glutaraldehyde-cetylpyridinium chloride.

Pollen from birch trees (Betula pendula) was fixed in glutaraldehyde containing 0.5% cetylpyridinium chloride (CPC), incubated with concanavalin A (Con A)-ferritin, postfixed in osmium, dehydrated, and embedded in Epon. On ultrathin sections, ferritin particles were observed closely associated with the electron-dense material precipitated by CPC on the surface of the pollen grains. Controls for CPC, which were fixed in glutaraldehyde alone, showed no electron-dense material on the surface. In controls for Con A, which were incubated in Con A-ferritin in the presence of the inhibitory sugar (alpha-methyl-D-mannopyranoside), no ferritin particles were observed. The above-described procedure thus allows the localization of sugar residues in highly soluble pollen wall glycoproteins.

Ultrastructural demonstration of a glycoproteinic surface coat in allergenic pollen grains by combined cetylpyridinium chloride precipitation and silver proteinate staining

SummaryIn allergenic birch pollen grains, highly watersoluble surface substances were precipitated by the cationic detergent cetylpyridinium chloride (CPC) during aqueous fixation. After processing the pollen for electron microscopy, ultrathin sections of pollen grains were subjected to the periodic acid — thiocarbohydrazide — silver proteinate (PA-TCH-SP) procedure according to Thiery (1967) for the detection of vicinal glycol groups. It was found that the material precipitated by CPC on the surface and within the exine cavities of the pollen wall strongly reacted with the PA-TCH-SP reagent thus indicating the presence of polysaccharides on the surface of birch pollen grains. In samples which had not been treated with the cationic detergent, PA-TCH-SP reactivity was reduced to thin linings on the surface and within the exine cavities. In both cases the exine proper did not stain whereas the intine showed moderate staining. Within the aperture region of the intine, PA-TCH-SP reactivity is preferably associated with fibrillar or reticular structures. The results are discussed with special reference to biochemical findings on allergenic birch pollen proteins.

Electron microscopic studies in cultivated plants

ZusammenfassungEs wurden raster- und transmissionselektronenmikroskopische Untersuchungen an der Hülse vonPhaseolus vulgaris var. nanus L. (Grüne Bohne) durchgeführt. Dabei lag der Schwerpunkt der Analyse bei dem Aufbau des Parenchymgewebes, welches den Hauptbestandteil der Hülse im eßbaren Zustand bildet. Der Blattcharakter der Hülse als Perikarp kommt besonders durch den Besitz von Stomata, Trichomen und typischen Cuticulastrukturen auf der Außenseite zum Ausdruck (SEM). Die Zellen des mesophyllartigen Parenchymgewebes zeichnen sich durch eine große morphologische Variationsbreite der Plastiden aus (Chloroplasten — Chloroamyloplasten — Amyloplasten) sowie durch einige spezielle cytologische Merkmale, wie ein stark entwickeltes rauhes ER, große Zellkerne und zahlreiche Plasmodesmen (TEM).SummaryScanning (SEM) and transmission (TEM) electron microscopic studies in the legumen ofPhaseolus vulgaris var. nanus L. (kidney beans) were carried out. In this work emphasis was laid on the analysis of the morphological structure of the parenchyma tissue, being the chief component of the pod at edible maturity. The leaf character of the pod becomes especially evident by the occurence of stomata, trichomes, and typical cuticle structures on the outside of the pod (SEM). The cells of the mesophyll-like parenchyma tissue are distinguished by a great range of variation of their plastids (chloroplasts — chloroamyloplasts — amyloplasts) and some special cytological features such as a strongly developed rough ER, big nuclei, and numerous plasmodesmata (TEM).

Immunoautoradiographic and protein-A/gold labelling experiments for localization of pollen allergens using antisera from atopic human individuals

SummaryUsing serum from human atopic individuals with a sufficiently high titre of IgE and IgG antibodies to birchor hazel-pollen allergens and antigens, the localization of IgE binding sites in birch- and hazel-pollen grain was determined by pre- and post-embedding electron microscopic immunoautoradiography with 125J-anti-IgE, whereas the IgG binding sites were localized in ultrathin sections of birch-pollen grains by the protein-A/gold technique. Concerning the distribution patterns of both IgE/IgG binding sites within the pollen grains, no difference could be observed in the dormant pollen grain: Labelling was found in the exine part of the pollen wall and throughout the highly condensed cytoplasm except for starch grains and lipid droplets. The intine part and the germination pores were almost completely unlabelled. In pollen grains which had been soaked in a hypotonic buffer for 15 min, however, IgE binding sites were predominantly localized within the intine and the germination pores. The specificity of the labelling reactions and the observed differences in the localization patterns are discused.

Electron microscopic studies in cultivated plants@@@Elektronenmikroskopische Studien an angebauten Pflanzen: II. Fresh and stored roots of daucus carota L.

ZusammenfassungEs wurden raster- und transmissionselektronenmikroskopische Untersuchungen an frischen und an 4 Monate bei durchschnittlich + 8° C gelagerten Wurzeln vonDaucus carota L. durchgeführt, wobei das sekundäre Phloemparenchym im Vordergrund der Analysen stand. REM-Aufnahmen zeigen die histologische Anordnung der einzelnen Zellen sowie die Umrisse von Stärkekörnern, Lipidtropfen, Mitochondrien und Carotinkristallen im cytoplasmatischen Wandbelag. Im TEM sind neben den normalen cytologischen Details (Mitochondrien, Golgi-Apparate, ER, usw.) Kerne sichtbar, die sich durch den Besitz mehrerer Kernkörperchen auszeichnen. Von großer morphologischer Vielfalt stellen sich Form und Struktur der Chromoplasten dar. Bei gelagerten Karotten kommt es zu einer starken Abnahme der Lipidtropfen sowie zu einem Stärkeabbau in den Chromoplasten.SummaryScanning (SEM) and transmission (TEM) electron microscopic studies in fresh and stored roots (4 months at an average temperature of + 8° C) ofDaucus carota L. (carrots) were carried out. Chief stress was laid on the analysis of the histological and cytological structure of the secondary phloem parenchyma cell. SEM images show the three-dimensional histological arrangement of the cells. Moreover, the outlines of lipid droplets, mitochondria, starch grains, and carotin pigment crystals are visible within the parietal cytoplasmic layer. In TEM, besides the usual cell organelles (mitochondria, golgi-apparatus, ER, etc.) nuclei with several nuclear bodies can be recognised.

Eichstandards für die quantitative Röntgenmikroanalyse biologischer Proben im Rastertransmissionselektronenmikroskop

Summary Quantitation in biological X-ray microanalysis usually depends on the reference to one or more element standards. This paper deals with the general characteristics of such standards and gives a survey of the various methods of standard preparation reported in literature. Their advantages and disadvantages are briefly discussed. Practical experiments were carried out to prepare a potassium, calcium, and sulfur standard for X-ray microanalysis in the scanning transmission electron microscope.

Electron microscopic morphometry of cell wall swelling in rehydrated carrots and green beans: The influence of various blanching, drying and storing parameters

ZusammenfassungMit Hilfe morphometrischer Messungen an transmissionselektronen-mikroskopischen Bildern wurde der Grad der Zellwandquellung bei rehydratisiertem Trockengemüse (Karotten und grünen Bohnen) in Abhängigkeit von verschiedenen Blanchier-, Trocknungs- und Lagerbedingungen analysiert. Die Ergebnisse lassen eine eindeutige Korrelation zwischen lebensmitteltechnologischen Verfahren und der Zellmorphologie der Proben erkennen.SummaryBy morphometric analysis of transmission electron-micrographs the degree of cell wall swelling in rehydrated vegetables (carrots and green beans) was examined as a function of various blanching, dehydration and storing parameters. The results show a clear correlation between food technology parameters and cellular morphology of the samples.