Glycoprotein Ib in the triton-insoluble (cytoskeletal) fraction of blood platelets

Glycoprotein Ib could be demonstrated in the Triton-insoluble (cytoskeletal) fraction of platelets prepared with EGTA by SDS-polyacrylamide gel electrophoresis and staining with the periodic acid Schiff's reagent. Crossed immunoelectrophoresis showed that glycoprotein Ib could be extracted from such Triton-insoluble residues when the extraction solution contained 1% Triton X-100 plus 5 mM CaCl₂, but not if it also contained leupeptin. This indicates that glycoprotein Ib was associated to structures in the cytoskeletal fraction in such a way that it could be extracted only after activation of a calcium-dependent protease, and degradation of the actin-binding protein was demonstrated. After crossed immunoelectrophoresis of platelet extracts prepared in the presence of leupeptin or EDTA, a glycoprotein Ib-related, rocket-shaped immunoprecipitate was seen originating from the application well. This was interpreted as being related to glycoprotein Ib associated to actin polymers which did not sediment at low-speed centrifugation. Incubation of platelets with ³²P as sodium phosphate led to incorporation of phosphatase-sensitive ³²P in all of the glycoprotein Ib-related immunoprecipitates except for that of glycocalicin. This support the idea that glycoprotein Ib traverses the plasma membrane and can be phosphorylated at the inner surface whereas glycocalicin represents the terminal part of the glycoprotein Ibα-chain exposed at the outer surface.     

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.     

An electron microscope study of the cell surface ofCytophaga johnsonii and some observations on related organisms

The cell surface of a non-fruiting myxobacterium,Cytophaga johnsonii, was investigated by electron optical techniques. Negative staining revealed an irregularly undulating surface topography and the presence of strands of material arising from the cells. Replicas of freeze-dried cells and thin sections confirmed the uneven nature of the surface and showed that, in contrast to eubacteria, the cell was surrounded by an envelope of amorphous, electron-translucent material contained within a crenated, outer, unit membrane. This material could be extruded as long strands. It is considered that this envelope of material represents the slime layer of the organism.The appearance of the slime layer at various stages in the growth of the organism was followed by negative staining and the image of the organism presented by negative staining was compared to that given byMyxococcus xanthus andFlavobacterium aquatile.     

Glycoprotein Ib beta is the only phosphorylated major membrane glycoprotein in human platelets.

Platelets were metabolically labelled with 32P and the phosphoproteins examined by two-dimensional non-reduced/reduced gel electrophoresis and isoelectric-focusing/gel electrophoresis. Comparison with similar separations of surface-labelled platelets showed that the only major glycoprotein which is phosphorylated is the beta-subunit of glycoprotein Ib, indicating that this subunit contains a cytoplasmic segment. The identification was confirmed using immunoblotting with an antibody to the beta-subunit. Phosphoserine was the principal phosphorylation site, with some phosphothreonine, but phosphotyrosine was absent. No quantitative or qualitative differences could be detected in the phosphorylation of glycoprotein Ib beta from resting or activated platelets. These results exclude changes in phosphorylation of the major platelet membrane glycoproteins as a method of signal transmission by these receptors.     

An electron microscope study of the perfusion-fixed spleen

Summary By preserving the intercellular or plasmatic space, perfusion fixation provides a particularly clear picture of the histological and cytological constitution of the spleen. An analysis of documents obtained with this technique has contributed new information concerning the organization of the intermediate circulation in that organ, and on the topographical relationships and ultrastructure of the various types of cell which are usually grouped under the common denomination of reticulo-endothelial system.Currently available physiological data concerning the flow of blood through the spleen, together with the observed scarcity of sinuses, and of interstices in the latters' walls, to which must be added the narrowness of these interstices, incline us to question the existence of a circulatory system involving the passage of blood from the arterioles into the pulp cords of Billroth, and thence into the sinuses. Thus we are led to conceive of a system whereby about 97% of the blood entering the spleen would flow directly into the sinuses. Phagocytosis of worn blood cells would be carried out by a quantitatively less important side-stream, running parallel to the main stream, but nevertheless sufficiently fast to ensure that every blood cell should be shunted through the spaces of Billroth at least once every 24 hours.The unitary concept of the RES, as it appears from the bulk of existing literature, is based upon the postulated, but never proven, existence of a single cell type capable of developing distinct morphological characteristics, and of carrying out distinct functions, namely phagocytosis and the laying down of reticulin, depending on its situation in the tissue. An ultrastructural study of the various cells encountered in sections of spleen, and a comparison with their counterparts in other organs, seem to rule out this unitarian view.

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Résumé La fixation par perfusion, en conservant l'espace intercellulaire ou plasmatique, donne une image de l'organisation structurale et cytologique de la rate particulièrement claire. L'analyse des documents aínsí obtenus fournit des données nouvelles sur l'organisation de la circulation intermédiaire et sur la position et l'aspect ultrastructural des cellules habituellement réunies sous le nom de système réticulo-endothélial. Les données de physiologie sur le débit splénique, le nombre de sinus observables et le relativement petit nombre et l'étroitesse des fentes sinusiennes font douter de l'existence d'un système circulatoire faisant passer le sang des artérioles dans les cordons de Billroth et de là dans les sinus. Ceci fait envisager un schéma de circulation faisant passer environ 97% du sang directement à travers le sinus. La phagocytose des éléments à détruire est assurée par une circulation parallèle peu importante, mais suffisante pour que chaque élément sanguin ait chaque jour la possibilité de passer par les cordons de Billroth.La théorie du SRE est basée sur l'existence d'une cellule unique, différant seulement par sa position et assurant la macrophagie et la formation de la réticuline. L'étude de ces différents types de cellules au niveau de la rate et leur comparaison au niveau d'autres organes permet d'assurer qu'aucun argument de morphologie ultrastructurale ne peut l'étayer.

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This work was supported by grant No 4237 of the Swiss National Foundation for Scientific Research.

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We wish to thank Mrs. Sidler-Ansermet for her invaluable technical assistance.     

An electron microscope study of myelin figures

In the electron microscope, thin sections of OsO(4)-fixed myelin figures from the phospholipide fraction of human brain show a pattern of parallel dark lines with a repeating period of about 40 A. It is shown that the dark lines probably represent the reaction product of OsO(4) with double bonds in the fatty acid chains, thereby marking the central portion of one bimolecular lamella. The addition of globin results in dense lines 25 to 50 A wide that cover the surface of the myelin figures. When such a figure consists of only two bimolecular leaflets of lipide covered with globin, the structure shows striking similarity to the image of cell membranes in fixed tissue sections. A hypothetical schema is given of the molecular structure of the figure, and the distribution of OsO(4) in it.     

An electron microscope study of lampbrush chromosomes

Lampbrush chromosomes were isolated from germinal vesicles of oocytes from Necturus maculatus, Triturus viridescens, Pseudotriton montanus and Rana pipiens. After treatment of isolated nuclei with 10 per cent sucrose, chromosomes free of nuclear sap are obtained for examination in either the light microscope or in the electron microscope. For electron microscopy the chromosomes were prepared either by Anderson's critical-point procedure or were embedded in methacrylate and sectioned. The evidence presented in favor of the view that the loops, axis, and the chromomeres of lampbrush chromosomes are formed by two chromonemata is based on the following observations: 1. Treatment of isolated chromosomes with 0.002 M KCN loosens the structure of the loops, and a more or less coiled organization is then observed in most of them with the light microscope. At the electron microscope level, each loop consists of a bundle of microfibrils. The latter are 500 A in diameter, and their complex arrangement within the loops is best studied in stereoscopic preparations. 2. Treatment of chromosomes with 0.002 M KCN also unravels the "chromomeric" regions of the axis. A fibrillar organization then becomes visible in the light microscope. In the electron microscope, wide strands are seen within some chromomeres; their diameter corresponds closely to that of the chromonemata forming the loops associated with the same chromomeres. In thin transverse sections of isolated chromosomes, no special structure is visible in the axial region except random profiles of fibrils similar to those seen in the loops of the same preparations. 3. Two strands sometimes connect adjacent chromomeres. Where gaps exist along the axis, after stretching of the chromosomes, a loop occasionally straddles the break and returns to a chromomere on each side.     

An electron microscope study on in vitro parthenogenesis in sunflower

Summary Electron microscope studies have been conducted on the parthenogenesis induced by in vitro culture of unfertilized ovules of sunflower (Helianthus annuus). In comparison with the state of the egg prior to inoculation, some eggs 5 days after culture show striking ultrastructural changes, which include, among others, nuclear migration, an increase in the number and activity of the organelles, a loss of polarity and wall formation at the chalazal end of the cell. Most of these changes are similar to those that occur normally in the zygote, indicating that parthenogenic development has been triggered in these eggs. Such eggs have been termed activated and are presumed to be capable of undergoing parthenogenesis. The parthenogenic proembryos which result share some features in common with zygotic proembryos. In addition, some parthenogenic proembryos exhibit unique properties not found in zygotic proembryos. These include embryos that consist of two parts differing markedly in density, an inversion of polarity, the frequent occurrence of autophagic vacuoles, the thickening of cell walls, a centripetal growth mode of wall formation, the appearance of an incomplete cell wall, free nuclear division, amitosis and degeneration. We believe that these ultrastructural peculiarities are the effects of in vitro culture.     

An electron microscope study of the yeast Pityrosporum ovale

Summary Cells of Pityrosporum ovale were prepared for electron microscopy by different methods of fixation and embedding, all of them causing some degree of damage to the cells. Apart from the usual organelles seen in other yeast cells, a body was found which showed an electron-dense outer layer and an electron-light centre when stained with permanganate. The cell wall showed layers of different electron-density. Buds were formed at one pole only, leaving a collar on the mother cell.     

An electron microscope study of the regenerating nerve fibers

Summary Guinea pig sciatic nerves were severed in order to obtain a regenerative process of the nerve fibers. The animals were killed at different periods of time after the severing (24 hours, 6 days and 12 days) and the specimens obtained were prepared for electronmicroscopic study.The nerve fiber growing extremities (growing cones) were specially studied. The growing cones showed the following components: a) microvesicles; b) mitochondria; c) multivesicular bodies.The microvesicles are hollow elements of about 200 to 700 Å. They constitute the main component of the growing cone. The mitochondria were seen as elongated bodies of 80 m. They were seen in many cases changing to round dense bodies which appear to break-up in irregularly-shaped fragments.The multivesicular bodies were found present in most of the growing cones. Protoneurofibrils do not exist in the growing cone but a close relationship between microvesicles and protoneurofibrils was found in the segments next to the growing cone.The above-mentioned components were found in all growing cones, disregarding the time elapsed after the nerve severing.Director of the Institute.Assistant of the Department of Neurohistology and Experimental Histology.Head of the Department of Cell Ultrastructure.