Lectin binding on carbohydrate compounds of the flask cells in the claw-frog kidney

The carbohydrate compounds of the mucus of flask cells in the kidney of claw-frogs (Xenopus laevis) were analysed through lectin binding studies. After removing epoxy resin semithin sections were incubated with 7 lectins (WGA, RCA I, PNA, LCH, UEA, LPA) marked by horseradish peroxidase and 2 unmarked lectins (VAA, Con A). The glycosaminoglycans in the canalicular lumen of flask cells showed a strong reaction with WGA and RCA, whereas the binding of PHA, Con A, and LCH was weaker. No reaction was observed with PNA, UEA, LPA, and VAA. The mucus of the flask cells seems to be rich in N-acetyl-glycosamine and -galactosamine. It contains also mannose, glucose, and galactose, but seems to have no fucose or N-acetyl-sialic acid residues.     

Freeze-fracture investigations of membranes of flask cells in the kidney and of parietal cells in the stomach of claw-frog (Xenopus laevis)

The flask cells of the nephron and the parietal cells in the stomach fundic glands of claw-frog show some structural similarities such as the existence of a canalicular system, the richness of mitochondria and numerous small cytoplasmatic vesicles. Both cell types also seem to have common functional pecularities. With the freeze-fracture method the cell membranes of both cell types showed a richness of intramembrane particles (IMPs) and the existence of special elongated IMPs especially in the apical and canalicular cell membranes. In the flask cells the elongated so-called rod-shaped IMPs measured 29 X 13 nm and were very numerous, whereas the corresponding particles in the parietal cells in the same membranes were smaller (13 X 5 nm) and not so numerous. The similarities and differences between the membranes of both cell types are discussed in relation to the cell function.     

Binding of the blood group-reactive lectins to human adult kidney specimens

The binding of a panel of blood group-reactive lectins to frozen sections of human kidney was studied with a special emphasis on reactivity with endothelia and basement membranes. The blood group A-reactive lectins, all specific for alpha-D-N-acetylgalactosamine (GalNAc), Helix aspersa (HAA), Helix pomatia (HPA), and Griffonia simplicifolia I-A4 (GSA-I-A4) agglutinins bound to the endothelium in specimens with blood groups A and AB. In other samples, these lectins reacted predominantly with tubular basement membranes, as well as with certain tubules. Both Dolichos biflorus (DBA) and Vicia villosa agglutinins (VVA), reported to react with blood group A1 substance, failed to reveal endothelia in most specimens, but bound differently to tubules in all blood groups. The blood group B-reactive lectins, specific for alpha-D-galactose (alpha-Gal) or GalNAc, respectively, GSA-I-B4 and Sophora japonica agglutinin (SJA), bound to the endothelia in specimens from blood group B or AB and in other specimens bound only to certain tubules. Among the blood group O-reactive lectins, specific for alpha-L-fucose (Fuc), Ulex europaeus I agglutinin (UEA-I) conjugates, but not other lectins with a similar nominal specificity, bound strongly to endothelia in specimens with blood group O. The UEA-I conjugates bound distinctly more faintly to endothelia in specimens of other blood groups. The present results indicate that lectins, binding to defined blood group determinants, react with endothelia in specimens of the respective blood group status. Furthermore, they suggest that basement membranes and some tubules in the human kidney show a distinct heterogeneity in their expression of saccharide residues, related to their blood group status.     

Localization of glycoconjugates at the tegument of the tapeworms Hymenolepis nana and H. microstoma with gold labelled lectins

Gold labelled lectins were used for electron microscopic localization of carbohydrate components of the tegument surface of two tapeworm species, Hymenolepis nana and H. microstoma. WGA, succinylated WGA, SBA, APA, PNA and, to a lesser extent, Con A were preferentially bound to the spines of the microtrichs. UEA-I and DBA were not adsorbed. The results indicate that the surface coat of both species has exposed N-acetylglucosamine, galactose and perhaps glucose and/or mannose residues.The location of lectin-binding glycoconjugates within the tegument and parenchyma was found using the light microscope on sections of material embedded in Lowikryl K4M after lectin-gold labelling and silver enhancement of the gold grains. The tegument selectively adsorbs WGA and SBA and strongly; adsorbtion of PNA and Con A is less intense. Strong adsorbtion of DBA and PNA was confined to the basal lamina. The parenchyma adsorbed Con A, PNA and DBA, but little WGA and SBA.The results indicate that many glycoconjugates are present in the tegument. They have similar terminal sugar residues to those of the surface coat. The significance of these carbohydrates for hostparasite interactions is discussed.     

Binding of lectins to Streptococcus mutans cells and type-specific polysaccharides, and effect on adherence.

The lectin concanavalin A (Con A) agglutinated the cells of 13 of 15 strains of the seven serotypes of Streptococcus mutans in an 18-h incubation period. Strains of types a, d, f, and g agglutinated within 2 h. Strains of a, d, and f were also agglutinated in 2 h by the castor bean lectin RCA. S. sanguis, S. salivarius, S. bovis, Actinomyces viscosus, A. naeslundii, and Lactobacillus plantarum were agglutinated within 2 h. The S. mutans type f polysaccharide was precipitated by Con A. The a, b, c, d, and e polysaccharides were not precipitated. Glucan from d and e strains of S. mutans and dextran T2000 were also precipitated by Con A. D-glucose inhibited the agglutination of type f cells by Con A and the agglutination of type d cells by D-galactose. The quantity of [acetyl-3H]Con A bound was not proportional to the degree of agglutination. Cells grown in sucrose medium bound more Con A than those grown in glucose medium. After treatment with dextranase, the sucrose-grown cells bound two- to fourfold more Con A. The binding of Con A to the type-specific polysaccharide or to teichoic acid could not be determined by the use of specific antibody due to the binding of Con A to the antibody globulin on the cell surface. Con A bound to S. mutans cells did not inhibit the activity of cell-bound glucosyltransferase, glucan synthesis, and in vitro adherence. Bound Con A also did not inhibit the ability of heat-treated cells to bind glucosyltransferase, synthesize glucan, and produce in vitro adherence.     

Binding and redistribution of lectins on lymphocyte membrane

The redistribution of fluorochrome-conjugated agglutinins bound to the lymphocyte plasma membrane is investigated. Two “T” lymphocyte mitogens (concanavalin A (Con A) and phytohemagglutinin (PHA)) bind to and redistribute equally well (spotting and capping) on the lymphocyte, whether they are “B” (Ig⁺) or “T” (Ig^?). One B cell mitogen (pokeweed mitogen (PWM)) does not bind detectably either to T or to B lymphocytes, although it binds to macrophages and to thymocytes in a perfectly ring-like pattern. Its redistribution requires anti-PWM antibodies. The nonmitogenic poly-L-lysine (PLL) binds to virtually all lymphocytes, and rapidly forms caps. PLL binds more strongly to T cells than to B cells. The agglutinin concentration is critical for the formation of agglutinin caps on the cell surface, as well as for the free mobility of Ig receptors. Finally, lectin-coated beads pick up B and T cells equally well, irrelevantly of their mitogenicity. Cell attachment to agglutinin-coated beads does not induce any demonstrable capping of either agglutinin-binding sites or membrane Ig. When the cells are further treated with soluble agglutinins or anti-Ig antibodies, they form caps mainly oriented towards the lectin-coated beads. These results show the high mobility of lymphocyte membrane components, the independence of agglutinin-binding sites and of most of the Ig receptors, and a polarization of cell with regard to cap formation. They exclude any straightforward correlations between agglutinin binding, membrane component redistribution and lymphocyte triggering.     

Effects of gold compounds on function of phagocytic cells

The effect of sodium aurothiomalate and auranofin on the generation of superoxide anions (O ₂ ^– ) by polymorphonuclear leukocytes (PMNLs) and adherent mononuclear phagocytic cells (AMNCs) has been investigated. Sodium aurothiomalate at final concentrations of 1, 10, and 100 g Au/ml and auranofin ranging from 0.1 to 2.0 g Au/ml were used in the reactions involving all celt types. Results have been compared between cells drawn from normal controls and patients with active rheumatoid disease. The effect of gold compounds on both cell types was assessed following activation by phorbyl myristate acetate (1×10^–8 M) and N-formyl-methionylleucyl-phenylalanine (1×10^–4 M) using a cytochromec reduction method. Sodium aurothiomalate at the maximum concentration modestly inhibited O ₂ ^– generation by PMNLs but not AMNCs. Auranofin inhibits O ₂ ^– generation by both cell types. Inhibition of cells from patients with rheumatoid arthritis was greater than that seen with cells from normal controls.     

Important role of the kidney in human carbohydrate metabolism.

Recent studies using a combination of isotope and balance techniques have shown that, in the postabsorptive state, the human kidney contributes substantially to overall glucose production and consumption. The kidney may contribute as much as the liver to gluconeogenesis and play an important role in the counterregulation of hypoglycemia. Furthermore, increased renal glucose production may contribute to fasting hyperglycemia found in type I and type II diabetes mellitus. Finally, loss of renal tissue as a consumer of glucose could explain the insulin resistance of uremia. We hypothesize that the human kidney may play a more important role in human carbohydrate metabolism than previously appreciated.     

Binding of the receptors for IgE by various lectins

Two receptors for IgE, having apparent molecular weights of 55,000 and 45,000 daltons were isolated from rat basophilic leukaemia cells by means of IgE-Sepharose. Both molecules were bound by concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin (castor bean lectin). The lectins of pea and gorse origin only bound the 45,000 dalton receptor.     

Effect of plant lectins on murine myeloma cells.

The mitogenic response (determined by uptake of [3H]-thymidine) of two different BALB/c myeloma lines, normal BALB/c spleen cells and spleen cells from tumour-bearing mice in primary culture to PHA, Con A, PWM and Robinia pseudoaccacia (RPA) extract was determined by measuring the uptake of [3H]-thymidine over 96 h. The pattern of the response of tumour and tumour-spleen cells to the 4 different lectins was similar, and different from that of normal spleen cells. The unstimulated cultures of tumour and tumour-spleen cells displayed an initial increased uptake of [3H]-thymidine, whereas stimulated cultures displayed a low initial uptake of label. After an initial phase of inhibition, ADJ-PC5 myeloma cells were stimulated by PHA and PWM to a greater extent than were normal spleen cells. On the other hand, normal spleen cells gave a markedly better response to Con A and RPA. The mitogenic response of tumour-spleen cells to all 4 lectins was intermediate between those observed for tumour and normal spleen cells; they responded better to Con A and RPA than did tumour cells but were not as responsive as spleen cells, whereas the converse was true for their response to PWM and PHA. In agreement with other reports, the data suggest that the responsiveness of tumour-spleen cells was due to the presence of tumour cells in this tissue. These results indicate that there is no definite evidence of an impaired lectin-responsiveness in lymphoid cells of mice bearing a myeloma.     

小鼠胚胎干细胞表面糖分子被凝集素特异性识别的研究

目的　探讨小鼠胚胎干细胞表面糖蛋白能够被特异性识别的凝集素。 方法　通过磁珠筛选出小鼠胚胎干细胞系ES-D3 和 ESC57BL/6中周期特异性胚胎抗原1(stage specific embryonic antigen-1,SSEA-1)阳性的胚胎干细胞,进行凝集素的特异性结合,流式细胞仪筛选和免疫荧光染色,观察凝集素特异性结合情况。 结果　磁珠法可以筛选出99%的SSEA-1阳性细胞,它们代表着未分化的胚胎干细胞。在SSEA-1阳性细胞中,凝集素的结合实验的流式筛选提示,凝集素DSL结合率达99%,多花紫藤凝集素(Wisteria Floribunda Lectin,WFL)的结合呈双相性,部分不结合,部分高度结合;而荆豆凝集素I( Ulex europaeus Agglutinin UEAI)几乎不结合。 结论　曼陀罗植物凝集素(Datura stramonium Lectin,DSL)可以象SSEA-1一样,作为小鼠胚胎干细胞的特异性标志物,而UEAI可以作为阴性标志物。     

Effects of plant lectins on virus growth in nonlymphoid cells.

Several plant lectins markedly reduce the numbers of virus plaque-forming units released by cultures of different cell types, treated either before or after infection. Antiviral activity, which is not due to activation of the interferon system, is long lived and is demonstrable against a variety of enveloped and nonenveloped cytocidal RNA and DNA viruses. Although several mechanisms are responsible for the total antiviral effect, depending upon the virus studied, major effects seem to be the blocking of virus release, agglutination of cell-associated as well as liberated virus, and, possibly, diminished production of virus.     

Binding of mitogenic plant lectins to human lymphocytes. Flow cytometric analysis

Several plant lectins, such as phytohaemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (ConA), Maclura pumifera (MPA) and Pisum sativum agglutinin (PSA), are potent mitogens for human lymphocytes. The pattern of activation induced, however, is not uniform for all mitogenic lectins. The different biological effects following lectin activation of human lymphocytes might be due at least in part to a differential binding of the various lectins to lymphocyte subsets. We have therefore studied the binding of five mitogenic plant lectins, namely PHA, PWM, ConA, MPA and PSA to three major human lymphocyte subsets as defined by anti-CD4, anti-CD8 and anti-CD16 monoclonal antibodies. Dual colour, flow cytometric analysis employing PE-conjugated monoclonal antibodies and FITC-conjugated lectins revealed that all subsets uniformly show high binding of PHA, whereas two different populations, one high binding and the other low binding, can be detected with PWM, ConA, MPA and PSA.     

Comparative studies on internalization of gold labelled mistletoe lectin I (MLI), its subunits, and of an immunotoxin into mouse L 1210V leukemia cells.

Using a preembedding electron microscopic technique, the binding and internalization of gold labelled mistletoe lectin I (MLI.Au), its 2 A subunits (MLI-A.Au) and of the B subunit (MLI-B.Au) in murine L 1210V leukemia cells was analysed. Furthermore, the endocytosis of a gold marked immunotoxin (MoAb-16-MLI-A.Au), consisting of a monoclonal antibody (MoAb-16) reacting with L 1210V cells and the cytotoxic A subunits (MLI-A) was detected. The cells were incubated with MLI.Au, MLI-A.Au, MLI-B.Au, or MoAb-16-MLI-A.Au at 37 degrees C for 1, 3, 5, 10, 20 or 30 min, respectively. Remarkable differences were found in the endocytotic pathway and internalization kinetics. The endocytosis of MLI, its subunits and of the immunotoxin has been compared to that of the other ligands in various systems.     

Abnormal fucosylation of ileal mucus in cystic fibrosis: I. A histochemical study using peroxidase labelled lectins.

Peroxidase conjugated lectins were used to analyse the glycoproteins of small intestinal mucins in normal infants and those with cystic fibrosis to ascertain whether there are any detectable histochemical differences in saccharide composition. A significant decrease in Lotus tetragonolobus (LTG) binding fucose was shown in normal small intestinal mucin starting around 36 weeks' gestation with total absence of staining at term and beyond. In contrast, the age matched patients with cystic fibrosis showed persistent and intense LTG binding of fucose. These results provide the first clear histochemical evidence that cystic fibrosis mucin is abnormal and confirm the findings of previous biochemical studies.     

Sequential immunofluorescence and infectivity studies on the replication of Herpesvirus saimiri in owl monkey kidney cells.

Methods are described for the preparation and authentication of a highly specific antiserum against herpesvirus saimiri (HVS) capsid antigens. The antiserum was used in immunofluorescence tests to follow the development of capsid antigens in HVS-infected owl monkey kidney cells throughout the virus replication cycle in parallel with sequential titrations of virus infectivity in both cells and medium. Fluorescence was detected as a round or oval, bright green area of staining at the centre of the nucleus which was similar in outline to the Cowdry type A inclusion seen in HVS-infected cells stained by haematoxylin and eosin. The first detection of fluorescence towards the end of the eclipse phase of the virus growth cycle, and its abolition by the treatment of infected cultures with cytosine arabinoside confirmed the identity of HVS capsid antigens as late antigens. The failure to detect fluorescence in the cytoplasm of HVS-infected cells has brought to light a conflict between the site of accumulation of virus capsid antigens as determined by immunofluorescence and the finding, by electron microscopy, of cytoplasmic immature particles in intact cells during the early stages of the virus replication cycle. The significance of this discrepancy is discussed in relation to its possible existence for other members of the herpesvirus group.     

Immunological studies of human placentae. Binding of complexed immunoglobulin by stromal endothelial cells.

Endothelial cells of foetal stem vessels in cryostat sections of normal, full-term human placentae bind fluorescein-conjugated heat-aggregated human IgG. Soluble immune complexes of sheep anti-human albumin also bind in a pattern that is similar to that of aggregated human IgG, but native human IgG is not bound by placental endothelial cells. Aggregated IgG binding, unlike soluble immune complexes, is blocked by pretreatment of the sections with antiserum to human IgM. Cross-blocking experiments with aggregated IgG and immune complexes suggest that they may be bound by different receptors.     

Detection of S-100 labelled cells in nasopharyngeal carcinoma.

S-100 antigen containing cells with dendritic features, recognisable by morphological and immunohistochemical criteria as belonging to the Langerhans' or interdigitating reticulum cell type, have been found in undifferentiated nasopharyngeal carcinoma. The presence of these cells, which have a special function of antigen presentation in immune responses, may be involved in a possible modulation of nasopharyngeal carcinoma associated Epstein-Barr virus infection and host-tumour interactions.