Fuzzy Coat Research Articles

Properties of plasma membranes from granulation tissue with reference to extracellular matrix.

Treatment with neuraminidase decreased the activity of Na+,K+-activated Mg2+-adenosine triphosphatase in plasma membranes isolated from experimental granulation tissue but not that of 5'-nucleotidase or leucine-beta-naphthylamidase. A temporary lowering of the pH of the plasma membrane suspension to 2-3 inactivated all three enzymes, which remained inactive after the pH had been readjusted to 7.4. Addition of dextran preparations to the membrane suspension decreased the activity of adenosine triphosphatase. Ethanol (0.4%) had a similar effect. These marker enzymes of plasma membranes were not affected by additions of hyaluronate, chondroitin sulfate, protein polysaccharide or soluble collagen. Serotonin stimulated the adenosine triphosphatase activity slightly. About 10-20% of the protein in the plasma membrane preparation was extracted with EDTA. This "fuzzy coat" fraction yielded a distinct gel-electrophoretic protein pattern. Hyaluronidase was not helpful in cleaving this surface layer from the plasma membranes.

Ultrastructure of Mutants of Streptococcus mutans with Reference to Agglutination, Adhesion, and Extracellular Polysaccharide

An electron microscopic study of S. mutans 6715-13 wild type and representatives of three distinct classes of glucan synthesis-defective mutants (which fail to form adherent microbial plaques but agglutinate normally in the presence of exogenous glucans) disclosed the presence of two sucrose-dependent, glucanase-sensitive, extracellular components. In the wild type, these extracellular glucans had predominantly fibrillar (with some globular) morphologies. However, in the mutant strains, there was a consistent reduction in or loss of the fibrillar components and dramatic increases in globular forms. A cell surface-associated fuzzy coat was consistently seen, and it was neither sucrose-dependent nor glucanase-sensitive. The data indicated that in vitro and in vivo adhesion and virulence at smooth tooth surfaces (all these properties dramatically reduced in the mutants) were causally and functionally related to the extracellular, fibrillar, glucan component, whereas in vitro glucan-mediated agglutination may be related to the cell-associated surface fuzzy coat.

Fine Structural Topochemistry of Entamoeba histolytica Schaudinn, 1903

The surface of Entamoeba histolytica trophozoites reared under cultural and in-vivo conditions was studied using cytochemical staining procedures: uranyl acetate, phosphotungstic acid, ruthenium red, concanavalin A, anionic or cationic colloidal iron, and poly-l-lysine. Morphologically, the glycocalyx of axenic E. histolytica differs from that of monoxenic and in-vivo trophozoites. The glycocalyx of axenic cells was more regular and compact than that of the latter. In monoxenic and in-vivo cells, the dense inner portion of the glycocalyx was overlaid by irregularly distributed peaks or more fibrillar material. The cytochemical data are consistent with the presence of an irregular mosaic of high electronegative charge densities, probably composed of acid mucopolysaccharides, glycoproteins, or glycolipids. The majority of charging seemed to be associated with the more filamentous portions of the glycocalyx. Perhaps the increased prevalence of these surface projections in the in-vivo cells is correlated with an increased resistance to host defenses and virulence. The accumulating body of evidence of the structure of the glycocalyx (reviewed by Winzler, 1970; Cook, 1968) indicates that this fuzzy cell coat is in direct continuity with or anchored within components of the plasma membrane and confers polarity upon the exterior surface of cells. Many roles have been proposed for the cell coat, including functions in permeability, antigenicity, cell-to-cell adhesion and interaction, recognition, phagocytosis, and enzymatic activity (Rambourg, 1971). Cell coats demonstrated by electron microscopy cannot, at present, be interpreted in precise biochemical terms. However, variations in staining affinities suggest that there are qualitative and quantitative differences between cell types (Martinez-Polomo, 1970). Such coats have been reported for various taxa of parasitic protozoa (Hampton and Rosario, 1966; Andreassen and Behnke, 1968; Ladda et al., 1969; Vickerman, 1969; Filadoro, 1969). Vickerman (1969) demonstrated the cyclical appearance of the cell coat during the life cycle of Trypanosoma rhodesiense as an adaptation to parasitism. Siddiqui and Rudzinska (1965) were first to report a fuzzy coat from culture forms of Received for publication 27 August 1973. * Supported, in part, by Research Grant AI-02347 from the NIH and Department of Defense Contract No. DADA 17-68-C-8023. tPresent address: Department of Biology, University of Georgia, Athens, Georgia 30601. Entamoeba invadens. El-Hashimi and Pittman (1970) reported its presence on trophozoites of E. histolytica from human infections but were unable to detect the structure on axenic trophozoites. Feria-Velasco et al. (1972) demonstrated a glycocalyx on axenic E. histolytica strain HK-9:NIH by staining with ruthenium red or alcian blue-lanthanum. The present study was undertaken to investigate the polyelectrolyte and cytochemical composition of the E. histolytica surface under both cultural and in-vivo conditions because of continued interest in surface activity associated with invasiveness. MATERIALS AND METHODS

Gamete Interaction During Fertilization inCampanularia—The Female Epithelial Cell Surface

Prior to fertilization in Campanularia flexuosa a capacitation-like interaction occurs between the spermatozoa and the epithelial cells that lead to and surround the eggs. Trypsin treatment of egg packets (eggs surrounded by epithelial cells) prevents the capacitation-like action and results in loss of fertilizability. Following treatment, the normal fuzzy surface coat of the epithelial cells is reduced from 200 A to 50 A. Even with increased amounts of sperm the fertilization rate of trypsin-treated eggs in the egg packets never reaches normal levels. This indicates loss of available epithelial cell surface interaction sites. Epithelial cell surface material is solubilized by incubation of egg packets in calcium- and magnesium-free sea water. Treatment of trypsinized egg packets with the solubilized material increases the fertilization rate. The possibility that the necessary capacitation-like interaction is mediated by the cell surface is discussed.

Ultrastructural topography of a trematode eggshell

Scanning electron micrographs show that the eggshell of the digenetic trematode, Cryptocotyle lingua, is a concavo-convex ellipsoid with a longitudinal ridge-like demarcation extending along the convex, but not the concave surface. Numerous apical villosities contribute to an undulating appearance of the shell surface. Transmission electron microscopy reveals a fuzzy coat on the shell surface. Cytoplasmic flaps protruding from the parasite's uterus appear to intertwine with the shell surface villosities.

Observations on the fine structure and peroxidase cytochemistry of normal rat liver Kupffer cells

Continuation of the investigation of sinusoidal lining cells in rat liver (120–124) has led to a description of the morphology and peroxidase cytochemistry of normal Kupffer cells. Kupffer cells were found to have a variable but often stellate shape and a surface fuzzy coat, visible after “direct” osmium fixation. Worm-like structures were restricted to Kupffer cells, as were the annulate lamellae. The dense bodies varied in shape, diameter, and density. Kupffer cells may rest on or be inserted in, but are not continuous with, the fenestrated endothelial lining (120). No gaps were observed between Kupffer cells and endothelial cells. These morphological features make it possible to distinguish the Kupffer cells from other sinusoidal lining cells. The Kupffer cells did not show fat droplets, glycogen particles, autophagic vacuoles, or multivesicular bodies. After incubation by either perfusion or immersion for the demonstration of peroxidatic activity in livers fixed for 40 seconds, only the Kupffer cells showed a positive reaction in the nuclear envelope, the RER, and annulate lamellae. This specific staining reaction therefore permits the light-microscopical recognition of Kupffer cells. Time-series incubation of Vibratome sections showed that the peroxidatic reaction in Kupffer cells is sensitive to the mode of fixation, H2O2, pH 9.0, 2 × 10−2 M AT, 10−2 M NaN3, boiling, and the omission of DAB or H2O2 from the medium. No significant retardation of the reaction occurred in the presence of 10−2 M KCN. It is concluded that a peroxidase rather than a catalase is responsible for the reaction with DAB.

Presence of antigen common to avion tumor viral envelope antigen in normal chick embryo cells

Antiserum against the envelope antigen of avian tumor virus of subgroup E was obtained from chickens bearing tumors induced by RSV(f). The capacity to absorb neutralizing activity from the antiserum was used to demonstrate the presence or absence of the envelope antigen in various types of cells. The specificity of the antigen was also confirmed by immuno-electron microscopy. Cell extracts or viable cells from uninfected chick embryos were generally positive for the envelope antigen when the embryos were also positive in both the formation of gs-antigen and helper activity. Chick embryo cells negative for these viral functions were also deficient in the envelope antigen. However, two exceptional embryos were found in which the amount of envelope antigen and the level of helper activity do not directly correlate with each other. The antigen common to that on the viral envelope appeared to be located on or or in the fuzzy coat at the cell surface.

Contact Digestion in Tapeworms

The activity of pancreatic a-amylase is increased in the presence of Hymenolepis diminuta, although the intact worms show no intrinsic amylolytic activity. Enhancement of amylase activity is a function of the number of worms present. Maximum relative enhancement occurs at very low enzyme concentrations, and there is an apparent increase in maximum velocity when substrate concentration is varied. The effect is readily reversible and is inhibited by polycations. Killed worms produce no enhancement of amylase activity. It is concluded that a surface adsorption is involved and the possible structural relationships are briefly discussed. The importance of differentiating intrinsic from adsorbed enzymes is also discussed. Previous studies of the brush border of the mammalian small intestine have shown that there are intrinsic, membrane-bound disaccharidases and phosphohydrolases in the outer cell surface (Crane, 1962; Newey et al., 1963). Similarly, the tapeworm Hymenolepis diminuta has been shown to have surface phosphohydrolases (Arme and Read, 1970; Dike and Read, 1971a, b). Further, the tapeworm enzyme seems to be spatially adjacent to the membrane system involved in glucose transport: The glucose product of glucose-6-phosphate hydrolysis has a kinetic advantage for absorption rather than for diffusion into the surrounding medium in vitro (Dike and Read, 1971b). In addition to intrinsic digestive enzymes, adsorbed digestive enzymes have been reported to be associated with the mammalian brush border. These are pancreatic in origin and have been referred to as contact digestive enzymes (Ugolev, 1965; De Laey, 1966a). Such enzymes are thought to be bound to the brush border fuzzy coat by electrostatic charge (De Laey, 1966b). Binding seems to depend on surface area available (De Laey, 1966c, 1967) and is readily reversible (Jesuitova et al., 1964). The kinetic parameters of pancreatic amylase are reported to change with adsorption to the brush border. However, some doubt has been cast on the conclusion that adsorption of pancreatic amylase to the mucosal surface enhances the action of the enzyme. McMichael and Dahlqvist (1968) and Alpers and Solin (1970) have shown that Received for publication 14 September 1971. * This work was supported by a grant from the NIH, U. S. Public Health Service (AI 01384). there are membrane-bound intestinal amylases in man and the rat. These are intrinsic to the mucosal surface and differ from pancreatic amylase in a number of respects. It has been pointed out that failure to recognize the presence of these membrane-bound amylases could lead to an erroneous conclusion that there is an enhanced activity of pancreatic amylase in the presence of mucosal tissue. Taylor and Thomas (1968) reported that the presence of tapeworms (three species) increased the rate of starch hydrolysis by bacterial a-amlyase in vitro and that the degree to which the enzyme was affected was related to worm surface area. The present study is an examination of this phenomenon, using pancreatic a-amylase and the rat tapeworm Hymenolepis diminuta. MATERIALS AND METHODS Hymenolepis diminuta was reared in the beetle Tenebrio molitor. Young male rats of the Holtzmann strain (Sprague-Dawley derived), weighing 100 to 120 g, were infected with 30 cysticercoids of H. diminuta. Ten or 11 days after infecting a group of rats, they were killed and worms flushed from the gut with a Ringer saline containing 25 mr tris-maleate buffer at pH 7.2 (KRT of Read et al., 1963). Worms from a group of rats were randomly distributed into samples, usually 15 worms per sample. Amylose hydrolysis was measured colorimetrically at 690 nm, using the iodine reagent described by Taylor and Thomas (1968). At this wavelength, the color obeys Beer's law between 10 and 120 /ug of amylose. A typical curve with varying concentrations of a-amylase is shown in Figure 1. Amylase was also determined by the method of Ujihara et al. (1965). To detect substrate hydrolysis, 3,5-dinitrosalicylic acid was added just prior to stopping the reaction by boiling. The absorbance was read at 520 nm. Standard

On the preparation and some properties of closed membrane vesicles from hog duodenal and jejunal brush border

Closed and nearly spherical vesicles were obtained from both hog duodenum and jejunum after mucosa homogenization in the absence of EDTA and a series of fractional centrifugations. The vesicles were found to contain large amounts of two of the characteristic enzyme markers of the brush border membrane (aminopeptidase and alkaline phosphatase). They were seen by electron microscopy on thin sections or after negative staining to be composed of an apparently intact, 90–100 Å-thick membrane overlaid by the fuzzy coat and to be partly filled by a fibrous material tentatively identified with the cross-filaments of the microvilli. This filling was not removed by 5 mM EDTA or/and 1 M Tris unless the structure of the vesicles was largely destroyed. Very few empty vesicles were obtained at the end of these treatments. The vesicles from hog duodenum and jejunum were observed to contain nearly 2 molecules of cholesterol for 1 molecule of phospolipids. Specific differences were noted between both types of vesicles at the level of their sugar composition and associated enzyme activities. For instance, the jejunal vesicles contained no sialic acid and no enterokinase. They contain, respectively, 2 and 4 times as much alkaline phosphatase and aminopeptidase as duodenal vesicles.

Role of bristle-coated membrane in the uptake of ferritin by rat macrophages

The distribution of horse ferritin in rat peritoneal macrophages was studied by electron microscopy. In the first few minutes after exposure of the cells to ferritin either in vivo or in vitro, the protein is found in invaginations of the surface membrane not yet isolated from the external medium. These caveolae are characterized by the presence of a prominent bristle coat on their cytoplasmic aspect and a less distinct fuzzy coat on the surface exposed to the exterior of the cell. At later stages ferritin is found in bristle-coated vesicles and later in larger smooth-surfaced vacuoles. Counts of bristle-coated membrane sites before and after exposure to ferritin suggest that ferritin is selectively bound to preformed patches of bristle-coated surface membrane which subsequently invaginate to form the coated vesicles. The question of possible induction of such sites by the presence of ferritin is raised but cannot be settled by the available data.

Osbservations on the Cysts of Entamoeba Histolytica

Trophozoites of the Laredo, Huff and W B strains of Entamoeba histolytica were grown in Nelson's encystment medium. When numbers of mature cysts were noted, the entire culture was fixed in buffered glutaraldehyde and post osmicated. Embedment was in Araldite and sections were viewed in a Hitachi HU 11-A microscope. With this method trophozoites and cysts in all stages of maturation were obtained.In the precyst, the first indication of the formation of the cyst wall is the appearance of a fuzzy coat on the plasmalemma of the rounded up trophozoite. Subsequently, exterior to the coat, a thick electron lucid area appears, which may contain debris from the culture medium.

Ultrastructure of Entamoeba Histolytica Trophozoites Obtained from the Colon and from in Vitro Cultures *

The structure of Entamoeba histolytica trophozoites obtained by aspiration from the human colon and by cultivation in vitro was examined with the electron microscope. Amebae from both sources contained poorly developed endoplasmic reticulum, numerous vacuoles, glycogen particles, small cylindrical electron-dense bodies, and crystalloid structures within the cytoplasm. A well-defined fuzzy coat was present on amebae from the colon. A specific mechanism for the extracellular release of enzymes was not observed. In cultures, a number of dead amebae, some engulfed by living amebae, were seen. It is postulated that lytic enzymes responsible for the pathogenic activity of E. histolytica are released only after the death of some amebae in the colony.

Morphogenesis of influenza A virus in Ehrlich ascites tumor cells as revealed by thin-sectioning and freeze-etching.

The budding of a tumor-adapted strain of influenza A(0) virus at the surface of Ehrlich ascites tumor cells was studied by electron microscopy. Thin sections of budding sites showed the formation of a fuzzy coat on the outside of the cell membrane and simultaneously the apposition of a dark layer on the inner side. The continuity of cellular and viral membrane seemed to be preserved up to the point where the virion remained attached by only a thin stalk. Freeze-etching of virus budding sites yielded pictures in which a clear differentiation between the viral membrane and the host cell membrane was visible. The breaks across the fuzzy coat revealed striations corresponding to the "spikes" seen in negative contrast, whereas tangentially broken virus particles were best interpreted by assuming that splitting occurred midway between the two outer layers of the envelope.

A comparative study of the concrement vacuole of certain endocommensal ciliates—a so-called mechanoreceptor

The concrement vacuole is a distinguishing cytoplasmic characteristic of ciliates of the families Buetschliidae and Paraisotrichidae. The vacuoles of the various species have some structural features in common; however, the distinctive morphologic qualities possessed by them permits their classification into two types: CV-1 (Buetschliidae) and CV-II (Paraisotrichidae). Two of the common features are: (a) oval concretions which give a positive reaction for calcium when subjected to the Alizarin red S test and which, in thin sections, display a laminated pattern, and (b) ciliary rootlets. The basic unit of each rootlet appears tubular and measures about 220–240 Å in diameter. These rootlets are derived from the kinetosomes of a group of presumably specialized cilia and together define a part (Paraisotrichidae) or all (Buetschliidae) of the vacuole's boundary. Speculation is offered concerning possible modes by which the concrement vacuole could function as a statoreceptor. The kinetosomes which constitute the somatic infraciliature are attached to a subtending fibrous band whose constituent filaments are 60–70 Å in diameter. This fibrous band, in addition to dividing the cytoplasm of both families into ectoplasm and endoplasm, partially encircles the CV-II type concrement vacuole. The cytopharyngeal armature is also composed of ciliary rootlets whose individual units are 220–240 Å in diameter. Together these units form a hexagonal pattern. There is no anatomical association between the components of the cytopharyngeal armature and the vacuole. The cytoplasm of organisms from both families contains gastrioles, a contractile vacuole, a large complement of rough variety of endoplasmic reticulum, and a host of vesicles and agranular cisternae. The plasma membrane is endowed with a fuzzy coat; it is particularly prominent on organisms of the family Buetschliidae. This coat is PAS-positive, a reaction which is not abolished by prior treatment with α -amylase.