Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry.

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.     

Ultrastructural features of acidic complex carbohydrates in the intercellular matrix of the ovarian follicles in adult mice

Summary In the intercellular matrix of the granulosa layer of the mouse ovarian follicles, ultrastructural features of acidic complex carbohydrates have been studied by means of dialyzed iron (DI) staining in combination with procedures of digestion with Streptomyces and testicular hyaluronidases. In the intercellular matrix, DI reactive structures containing acidic complex carbohydrates consist of layers of a variable thickness coating the plasma membrane of the granulosa cells and reticular elements distributed in the spaces between the cells. The latter exists in two appearances; one is clumped masses of irregular shapes and different sizes, whereas the other being filamentous figures radiating from the masses. The effects of digestion with Streptomyces and testicular hyaluronidases upon the DI staining of the tissues indicate that the DI reactive structures in the intercellular matrix contain at least three types of acidic complex carbohydrates; hyaluronic acid, isomeric chondroitin sulfates and other acidic glycosaminoglycans. The histophysiological activities played by these particular complex carbohydrates have been briefly discussed.     

Ultrastructural features of acidic complex carbohydrates in the intercellular matrix of the ovarian follicles in adult mice.

In the intercellular matrix of the granulosa layer of the mouse ovarian follicles, ultrastructural features of acidic complex carbohydrates have been studied by means of dialyzed iron (DI) staining in combination with procedures of digestion with Streptomyces and testicular hyaluronidases. In the intercellular matrix, DI reactive structures containing acidic complex carbohydrates consist of layers of a variable thickness coating the plasma membrane of the granulosa cells and reticular elements distributed in the spaces between the cells. The latter exists in two appearances; one is clumped masses of irregular shapes and different sizes, whereas the other being filamentous figures radiating from the masses. The effects of digestion with Streptomyces and testicular hyaluronidases upon the DI staining of the tissues indicate that the DI reactive structures in the intercellular matrix contain at least three types of acidic complex carbohydrates; hyaluronic acid, isomeric chondroitin sulfates and other acidic glycosaminoglycans. The histophysiological activities played by these particular complex carbohydrates have been briefly discussed.     

Involvement of vesicle coat material in casein secretion and surface regeneration

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces.     

Localization and characterization of carbohydrates in adrenal medullary cells

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.     

Recognition of foreignness by blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat

The structure and function of the cell coat of the blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were studied with ultrahistochemical tests, including concanavalin A (Con A) labeling, and with in vitro phagocytosis experiments. The cell coat is intensely stained by ruthenium red and tannic acid. The cells possess binding sites for Con A. Proteolytic enzymes destroy the receptors for Con A and totally inhibit the phagocytic activity of amoebocytes. Incubation experiments with proteases, carbohydrases, and inhibition sugars revealed that (1) the Con A binding sites are anchored in the plasma membrane by proteins, and (2) glucose, fructose, mannose, and to a lesser extent N-acetylglucosamine and N-acetylgalactosamine, inhibit the binding of Con A to amoebocytes, suggesting that these carbohydrates might form part of these binding sites.     

THE ENTERIC SURFACE COAT ON CAT INTESTINAL MICROVILLI

The enteric microvilli of the cat, bat, and man are coated with a conspicuous layer composed of fine filaments radiating from the outer dense leaflet of the plasma membrane. This surface coat is prominent on the absorptive cells but is not so thick on the goblet and undifferentiated crypt cells. In other species the surface coat is poorly developed or inconsistent, but all intestinal microvilli have traces of such a coating over the tips and sides of the microvilli. Tissues prepared by the ordinary sectioning techniques for electron microscopy usually reveal this component when stained with uranyl acetate followed by lead staining. The surface coat is intensely periodic acid-Schiff (PAS) positive and reacts with Alcian blue or Hale's colloidal iron stain for acid mucopolysaccharide. It is also stained by toluidine blue at low pH. Repeated washings or incubation with various chemical agents have failed to remove or markedly alter the appearance of the coating, but extruded cells undergoing autolysis lose their surface coats. The stability, consistent presence, and intimate association of the mucopolysaccharide coat suggest that it may be an integral part of the plasmalemma rather than an "extraneous coat."     

Rapid lateral diffusion of the variant surface glycoprotein in the coat of Trypanosoma brucei

The membrane form of the variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of Trypanosoma brucei by a dimyristoylphosphatidylinositol residue connected via a glycan to the COOH-terminal amino acid. The glycoprotein molecules are tightly packed, forming a coat that is impenetrable to lytic serum components. Lateral diffusion of mfVSG was measured by the fluorescence recovery after photobleaching technique. mfVSG labeled on the cell surface with rhodamine-conjugated anti-VSG Fab fragments showed a diffusion coefficient of 1 X 10(-10) cm2/s at 37 degrees C and of 0.7 X 10(-10) cm2/s at 27 degrees C. About 80% of the molecules were mobile. Affinity-purified mfVSG molecules implanted into the plasma membrane of baby hamster kidney cells exhibited a similar mobility to that found in the trypanosome coat [D = (0.4-0.7) X 10(-10) cm2/s at 4 degrees C]. Phospholipid mobility in the plasma membrane of trypanosomes was characterized by a diffusion coefficient of 2.2 X 10(-9) cm2/s at 37 degrees C. It is concluded that mfVSG mobility in the surface coat of the parasite is rapid and comparable to that of other membrane-bound glycoproteins but slower than that of phospholipids.     

Electron microscopic study of glomerular filtration barrier in the rat kidney embedded in polymerized glutaraldehyde-urea.

Embedding kidney in polymerized glutaraldehyde-urea favors the retention of glycoprotein matrix of the cell coat and the basement membrane of the glomeruli. The basement membrane appears as a single layer with uniform amorphous matrix. Thick glycoprotein coat covers the whole surface of prodocytes and their foot processes. In areas other than the slits and the portion of the foot processes which touch on the basement membrane, the coat is a continuous layer with an average thickness of 490 A. In the slits between the foot processes of podocytes there is an actual fusion of glycoprotein coats; the average width of the slit is 415 A. The glycoprotein 'plugs' in the slit may be a significant portion of the glomerular filtration barrier against macromolecules, together with the basement membrane and the slit diaphragms.     

Localisation of the MRC OX-2 Glycoprotein on the Surfaces of Neurones

The MRC OX-2 monoclonal antibody recognises membrane glycoproteins of Mr 41,000 in rat brain and 47,000 on thymocytes. It also reacts with follicular dendritic cells in lymphoid organs, endothelium, smooth muscle, and B-lymphocytes. Indirect immunoperoxidase staining of cryostat sections showed that OX-2 antigen was present throughout the cerebellum, with staining of both grey and white matter. Blood vessels were also stained. The Purkinje cell layer appeared to be unlabelled. Double-immunofluorescence staining of cerebellar interneurone cultures with MRC OX-2 antibody and tetanus toxin showed that all tetanus-positive cells (neurones) were MRC OX-2-positive. Glial fibrillary acidic protein-positive astrocytes were not labelled by MRC OX-2 antibody. Thus OX-2 antigen is one of the few biochemically characterised components of neuronal membranes and its properties are compared with those of the neuronal membrane glycoprotein Thy-1.     

Involvement of bristle coat structures in surface membrane formations and membrane interactions during coenocytotomic cleavage in caps of Acetabularia mediterranea

In the multinucleate cap rays of the green alga Acetabularia mediterranea the cell surface increases dramatically within a short time period during the final stages of coenocytotomic cleavage. In early stages of cyst formation the cytoplast is traversed by numerous large and prolate cleavage vesicles which are characterized by typical columellar or spinous coat structures. The cleavage vesicles are closely associated with the surface of plastids and, to a lesser degree, of mitochondria. This intimate association seems to be mediated by regularly spaced, densely stained intermembranous cross-bridge structures and is maintained throughout cleavage. These cleavage vesicles contain a finely fibrillar material structurally similar to the hyaline layer of mucilage that fills the space between the plasma membrane and cell wall. They line up with invaginations of the plasmalemma and vacuole membranes and, together with smaller vesicles interspersed, constitute preformed "perforation lines" for the final separation of the coenoblast portions. Equidistantly spaced plaques of attachment of such vesicles with surface membrane are described. We hypothesize (a) that the cleavage vesicle membrane is the immediate precursor to the new postcoenocytotomic surface membrane, (b) that the cleavage vesicle coat structures are integrated into the subsurface coat of the plasma membrane, (c) that growth of the laterally attached cleavage vesicles by intussusception of small fuzzy-coated vesicles is confined to their "free ends," (d) that the intermembranous cross- bridge elements are related to bristle coat structures and play a role in the establishment of the cleavage lines, and (e) that the coenocytotomic cleavage process is organized so that adjacent plastids are separated in a way that guarantees the inclusion of several plastids in each cyst.     

Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radioautography after labelled fucose injection into rats

A single intravenous injection of L-[³H]fucose, a specific glycoprotein precursor, was given to young 35–45 g rats which were sacrificed at times varying between 2 min and 30 h later. Radioautography of over 50 cell types, including renewing and nonrenewing cells, was carried out for light and electron microscope study. At early time intervals (2–10 min after injection), light microscope radioautography showed a reaction over nearly all cells investigated in the form of a discrete clump of silver grains over the Golgi region. This reaction varied in intensity and duration from cell type to cell type. Electron microscope radioautographs of duodenal villus columnar cells and kidney proximal and distal tubule cells at early time intervals revealed that the silver grains were restricted to Golgi saccules. These observations are interpreted to mean that glycoproteins undergoing synthesis incorporate fucose in the saccules of the Golgi apparatus. Since fucose occurs as a terminal residue in the carbohydrate side chains of glycoproteins, the Golgi saccules would be the site of completion of synthesis of these side chains. At later time intervals, light and electron microscope radioautography demonstrated a decrease in the reaction intensity of the Golgi region, while reactions appeared over other parts of the cells: lysosomes, secretory material, and plasma membrane. The intensity of the reactions observed over the plasma membrane varied considerably in various cell types; furthermore the reactions were restricted to the apical surface in some types, but extended to the whole surface in others. Since the plasma membrane is covered by a "cell coat" composed of the carbohydrate-rich portions of membrane glycoproteins, it is concluded that newly formed glycoproteins, after acquiring fucose in the Golgi apparatus, migrate to the cell surface to contribute to the cell coat. This contribution implies turnover of cell coat glycoproteins, at least in nonrenewing cell types, such as those of kidney tubules. In the young cells of renewing populations, e.g. those of gastro-intestinal epithelia, the new glycoproteins seem to contribute to the growth as well as the turnover of the cell coat. The differences in reactivity among different cell types and cell surfaces imply considerable differences in the turnover rates of the cell coats.     

Ultrastructural aspects of the surface coatings of eggs and larvae of the starfish,Pisaster ochraceus,revealed by alcian blue

Eggs of the asteroid Pisaster ochraceus demonstrate cortical granules, a thick vitelline membrane, and a poorly stained jelly coat similar to that seen on the eggs of other echinoderms. When fixed in the presence of alcian blue the jelly coat is seen to be made up of three regions, an inner layer consisting of a meshwork of fibres, a middle layer of thicker fibres, and a dense outer layer. At fertilization the cortical granules release their contents into the potential space between the vitelline layers and a low fertilization membrane consisting of the vitelline layer and a dense component of the corticle granule is formed. Initially the remaining contents of the corticle granules form an amorphous hyaline layer that fills the space between the plasma membrane and the fertilization membrane. At hatching a distinct hyaline layer is present. It persists at least to the bipinnaria stage and consists of four distinct layers. A similar layer is also located over much of the early embryonic endoderm but is lost from the regions involved in the formation of the mesenchyme cells, coelom, and mouth just before these events take place. Numerous large clear vesicles are located in the apex of all cells associated with a hyaline layer. Where the hyaline layer is lacking, only scattered vesicles are present suggesting that the vesicles may be involved in maintenance of the layer. Attempts to identify elements of the hyaline layer by immunofluorescence demonstrated that it appears to bind both antisera and control sera in a nonspecific manner.     

Human fetuin/α2 HS glycoprotein in colloid and parenchymal cells in human fetal pituitary gland

An immunohistochemical study was undertaken, in an attempt to identify the acidic glycoprotein(s) present in colloid and in parenchymal cells in human fetal pituitary gland. As the colloid has been proposed to represent disintegrating cells, a series of antibodies against plasma glycoproteins and plasma proteins was applied; their presence intracellularly would generally be an indicator of plasma membrane leakage in dying parenchymal cells. In tissue sections from 9- to 20-week-old fetuses, the colloid showed prominent staining with an antibody to human fetuin/₂ HS glycoprotein. Anti-₂-HS glycoprotein labelled parenchymal cells in pars anterior and intermedia. Apart from a minor immunoreactivity for ₁ glycoprotein, no other plasma glycoprotein was seen in colloid or parenchymal cells. An antibody against bovine fetuin showed staining of the colloid and of some parenchymal cells in pars distalis and intermedia; the plasma and stroma of the pituitary gland were unstained. In contrast, the anti-human plasma protein antibodies all stained the stroma. The presence of ₂ HS glycoprotein in parenchymal cells and absence of other plasma glycoproteins imply integrity of the parenchymal cell plasma membrane. Thus, ₂ HS glycoprotein is either synthesized locally or taken up specifically in the parenchymal cells, which are proposed to participate in the formation of colloid. It is suggested that ₂ HS glycoprotein is part of a homeostatic system, which controls remodelling and physiological cell death during development.     

Initial attachment of baby hamster kidney cells to an epoxy substratum. Ultrastructural analysis

In the presence of serum-containing medium, BHK cells attached and spread during a 1-h period onto a 3-5 nm thick serum layer absorbed on the substratum surface. The closest approach of the plasma membrane to the serum layer was observed to be about 9nm, which was determined by tilting the sectioned cells in a goniometer holder. Bundles of microfilaments or other cytoplasmic specializations were not observed in association with the regions of close contact. However, in the space between the plasma membrane and the adsorbed serum layer, a diffusely stained material could be visualized after fixation/staining by the tannic acid-glutaraldehyde technique. This technique also permitted increased clarity of visualization of trilaminar appearance of the plasma membrane. The distribution and mobility of anionic sites on the surfaces of attached and spreading cells was determined by labeling with polycationic ferritin. We observed movement of polycationic ferritin into large clusters on the cell surface, collapse of cell surface microextensions, and endocytosis, all of which were similar to our previous findings utilizing cells in suspension. However, the absolute amount of ferritin bound to the upper cell surface was less than that previously observed when suspended cells were put under similar labeling conditions. Also, polycationic ferritin did not appear to penetrate between the lower cell surface and the substratum.     

Ultrastructure of Cell Envelopes of Bacteria of the Bovine Rumen

Most of the bacteria found in rumen fluid samples taken from cows fed hay, or a concentrate diet, had cell walls of the gram-negative type. Most were intact, with only a small proportion of lysed cells, and many of the cells contained electron-translucent cytoplasmic deposits similar to the carbohydrate reserve material described in pure cultures of rumen organisms. All of the bacteria observed in these samples had an external "coat" layer outside the outer membrane when fixed in glutaraldehyde and osmium, stained with uranyl acetate and lead citrate, and examined as sectioned material. These coat layers varied from thin (ca. 8 nm) structures to very extensive fibrous systems, sometimes including concentric arrangements and radial fibers extending up to 1,200 nm from the cell. The thin-coat layers sometimes exhibited a rough periodicity. In all, 10 different types of coat layers were distinguishable on a morphological basis. It is proposed that these external coat layers have protective and adherence functions for the rumen bacteria in the environment.     

Lectin staining patterns of plasma membranes of daunorubicin and vincristine resistant Ehrlich ascites tumour cells

The plasma membrane is considered to play a major role in the development of resistance to anthracycline and vinca alkaloid drugs (pleiotropic resistance). Previous studies have reported an increase in plasma membrane carbohydrates in pleiotropic resistant cells compared with wild-type cells. The present study has utilized a panel of 11 lectins and the streptavidin-biotin histochemical technique in order to compare plasma membrane carbohydrates from wild-type Ehrlich ascites tumour cells with cells from daunorubicin and vincristine resistant sublines. While the lectins ConA, LCA, PSA, PNA after neuraminidase and WGA stained plasma membranes of daunorubicin-resistant cells to a significantly greater degree than those of wild-type cells, no difference was apparent between vincristine-resistant and wild-type cells. PWM and WGA after neuraminidase pretreatment showed similar staining of the wild-type and both resistant sublines, while SBA with and without neuraminidase pretreatment, HPA, DBA, LTA and UEA I demonstrated either very weak or negative reactions with all sublines. We conclude that the observed increase in plasma membrane carbohydrate found in anthracycline-resistant cells is possibly due to drug action during acquisition and maintainance of resistance, and, though conceivably of importance in the development of resistance towards anthracyclines, is without significance for the pleiotropic resistance phenotype itself.     

Focal cell contacts detected by ruthenium red, triton X100 and saponin in the granulosa cells of mouse ovary

Granulosa cells in growing follicles of mouse ovary, observed after treatment with ruthenium red (RR) as described by Luft (1971a, b), appeared to be covered by a continuous well-defined layer. On the contrary, treating granulosa cells with 1% Triton X100 (Vaccaro and Brody, 1981), followed by RR staining, resulted in the complete extraction of the plasma membrane coat (Triton does not affect the basement membrane and extracellular matrix proteoglycans). The use of 0.02% saponin together, with the RR stain, or 0.1% Triton X100 followed by RR staining, allows good visualization of follicular basement membrane and extracellular matrix proteoglycans without destroying cell morphology. Using this technique, we observed the extraction of the plasma membrane coat, but focal RR-stained condensations that were unaffected by saponin or 0.1% Triton X100 treatment were observed between plasma membranes of granulosa cells located around the periphery of large Graafian follicles. In some cases, RR condensations were located at the apex of plasmalemmal evaginations, in proximity to adjacent granulosa cells. Focal condensations of RR stain were never observed in secondary follicles. Present evidence suggests that focal cell contacts are mediated by transmembrane intercalated glycoproteins or proteoglycans and consequently play a role in cell adhesion. Their presence among granulosa cells of only very large Graafian follicles may be related to the maturation process of granulosa cells.     

DETECTION OF COMPLEX CARBOHYDRATES IN THE GOLGI APPARATUS OF RAT CELLS

Two methods used for the electron microscopic detection of glycoproteins were applied to a variety of cell types in the rat; one involved successive treatment of sections with periodic acid, chromic acid, and silver methenamine; and the other, a brief treatment with a chromic acid-phosphotungstic acid mixture. The results obtained with the two methods were identical and, whenever the comparison was possible, similar to those obtained with the periodic acid-Schiff technique of light microscopy. In secretory as well as in nonsecretory cells, parts of the Golgi apparatus are stained. The last saccule on one side of each Golgi stack is strongly reactive (mature face), and the last saccule on the other side shows little or no reactivity (immature face); a gradient of reactivity occurs in between these saccules. The more likely explanation of the increase in staining intensity is that carbohydrate is synthesized and accumulates in saccules as they migrate toward the mature face. In many secretory cells, the mature face is associated with strongly stained secretory granules. Other structures stained are: (1) small vesicles, dense and multivesicular bodies, at least some of which are presumed to be lysosomal in nature; (2) cell coat; and (3) basement membrane. The evidence suggests that the Golgi saccules provide glycoproteins not only for secretion, but also for the needs of the lysosomal system as well as for incorporation into the cell coat and perhaps basement membrane.     

Surface coat in steroid-secreting cells of the mouse ovary

Steroid-secreting cells (luteal, thecal and interstitial cells) of the mouse ovary have been studied with the ruthenium red method to stain the "cell coat". The results showed that a typical cell coat covers the entire surface of the plasma membrane except where the cells are connected by specific cell junctions. Further, particularly heavy concentration of ruthenium-red-reacting material was demonstrated in pericapillary and intercellular spaces. The differences in the thickness of the cell coat and its topographical modifications among different groups of steroidogenic cells may be related not only to intercellular adhesion and interactions but also concerned with a function of control in which the cell permeability is modified in relation with phenomena of cell recognition.