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Biomedical subjects

N Sharon

Publications and source records attributed to N Sharon.

At least 127 records · Page 7Linked to original sources
PubMed1984

Carbohydrates as recognition determinants in phagocytosis and in lectin-mediated killing of target cells.

Carbohydrate-lectin interactions serve as the basis of recognition by phagocytic cells of particles and of various target cells. Such interactions occur in the following systems: between sugars on the surface of the phagocytic cells and lectins on the surface of other cells--the best studied example is the binding of mannose-specific Escherichia coli and related organisms via their surface lectins to oligo-mannose residues on macrophages; between lectins on the surface of phagocytic cells and sugars on particles or other cells--phagocytosis of zymosan and of sialidase-treated erythrocytes, mediated respectively by mannose-specific and galactose-specific lectins on macrophages, belongs to this category; by extracellular lectins that form bridges between sugars on both types of cell--as shown by enhancement of phagocytosis of staphylococci by wheat germ agglutinin, and by lectin-dependent killing of target cells by macrophages. These interactions may play an important role in the activities of phagocytic cells in vivo. They may provide an initial host defense mechanism immediately after microbial infection, operate in tissues where phagocytic activity is poor, and participate in tumor rejection.

Animals↗
PubMed1984

Recognitory bacterial surface lectins which mediate its mannose-specific adherence to eukaryotic cells.

Cell surface protein were found to play a role in the sugar-specific molecular mechanism by which bacteria adhere to mammalian cells. We have demonstrated that at least three different types of lectin-like proteins mediate the mannose-sensitive adherence of gram negative bacteria to epithelial cells. One group of such lectins was shown in our study to be associated with the bacterial flagellum. Flagella isolated from Escherichia coli 7343 and Serratia marcescens 8347 exhibited mannose-sensitive agglutination of yeast cells; however, the flagella of the two bacteria differ in the molecular structure of their protein subunits. Another class of lectins comprises the bacterial fimbriae (also known as type 1 pili), which were previously shown to facilitate the mannose-sensitive adherence of various bacteria to mammalian cells. Fimbriae isolated from E. coli 346 were reversibly dissociated by saturated guanidine hydrochloride to their protein subunits. The dissociated subunits retained in part their mannose-binding ability, and were reassembled into fimbriae-like structures by removal of the denaturant under specific conditions. Mannose-sensitive yeast agglutinating activity of E. coli 2699, as well as of its isolated outer membranes devoid of fimbriae or flagella, was abolished by pretreatment with trypsin. It is therefore believed that the mannose-sensitive adherence of these bacteria is mediated also by lectin-like proteins associated directly with the outer membrane.

Animals↗
PubMed1984

Carbohydrate-binding sites of the mannose-specific fimbrial lectins of enterobacteria.

The combining sites of type 1 fimbrial lectins of various species of enterobacteria were studied by measuring the inhibitory activity of linear and branched oligosaccharides and several glycosides of D-mannose on the agglutination of yeast cells by the organisms. The results showed that all five strains of Escherichia coli tested possessed an elongated combining site best fitting a trisaccharide and including a hydrophobic region. Similar results were obtained with Klebsiella pneumoniae. Within the Salmonella genus, the combining sites of the six species tested were similar, but all differed significantly from those of the E. coli strains. The combining sites of Enterobacter cloacae and Enterobacter agglomerans were different from each other and from those of Salmonella sp. and E. coli. The results suggest that although classified under the general term "mannose-specific," bacterial lectins in the form of type 1 fimbriae on different genera exhibit differences in sugar specificities.

Binding, Competitive↗
PubMed1984

Wheat germ agglutinin potentiates uptake of bacteria by murine peritoneal macrophages.

Exposure of thioglycollate-elicited murine peritoneal macrophages to wheat germ agglutinin (WGA) increased markedly the uptake of six different bacteria, which have surface receptors for the lectin. Uptake of Staphylococcus aureus H was higher by 3-5-fold, of S. aureus 52A2 by 1.8-fold, of S. aureus 52A5 by 1.7-fold, of S. albus by 2.3-fold, of Shigella flexneri by 6-fold and of Micrococcus luteus by 6.5-fold. Klebsiella pneumoniae, devoid of receptors for WGA, was not phagocytosed following pretreatment of macrophages with the lectin. Pretreatment of the bacteria with the lectin also resulted, in most cases, in an increase in phagocytosis. Interaction of WGA with the macrophages and with the bacteria, as well as the potentiation of phagocytosis, was abolished by tri-N-acetylchitotriose, a saccharide that binds specifically to WGA, but not by monosaccharides which do not interact with this lectin. With non-elicited macrophages, enhancement of phagocytosis by WGA was less pronounced, probably because of the higher number of lectin-binding sites (5-fold) on the elicited cells. Peanut agglutinin and soybean agglutinin, that bind to macrophages but not to the bacteria studied, lack the ability to potentiate phagocytosis. Macrophage surface sugars thus appear to play an important role in phagocytosis by serving as receptors for lectins that form bridges between the macrophages and the microorganisms.

Animals↗
PubMed1983

Dynamic evidence for an extended subsite structure of the ligand combining site on wheat germ agglutinin: temperature-jump relaxation with fluorescence detection.

Temperature-jump relaxation methods have been used to study the binding kinetics of fluorescent 4-methylumbelliferyl glycosides of N-acetyl-beta-D-glucosamine and its beta (1 leads to 4)-linked di- and trisaccharides with wheat germ agglutinin. The mono- and disaccharide derivatives yielded biexponential progress curves. The data are consistent with two simple mechanisms in which binding occurs to an extended combining site on the lectin, consisting of at least two different, mutually exclusive, binding subsites. For one model, the bound ligand must slide from one subsite to the other, and the other mechanism requires the dissociation of the bound ligand from the protein before it can combine to the other subsite. Binding of 4-methylumbelliferyl monosaccharide to nonequivalent sites is improbable. The underlying kinetic and equilibrium parameters were obtained for the proposed subsites. The binding kinetics of the 4-methylumbelliferyl trisaccharide derivative are more complicated and may result from ligand-mediated linking reactions between molecules of the lectin. This study emphasizes that binding studies at equilibrium should take into account that the data result from an average of different binding configurations of all the ligands.

Acetylglucosamine↗
PubMed1983

Carbohydrate specificity of the surface lectins of Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium.

A large number of linear and branched oligosaccharides and several glycosides of D-mannose were tested for their inhibitory activity on the agglutination of yeast cells or guinea pig erythrocytes by three D-mannose-specific enteric bacteria possessing type 1 fimbriae. With Escherichia coli 346, the best inhibitors found are the alpha glycosides of the branched oligosaccharides alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-alpha-D-Manp-(1 leads to 6)-alpha-D-Manp-(1 leads to 3)-D-Manp and alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-alpha-D-Manp- (1 leads to 6)-[alpha-D-Manp-(1 leads to 2)-alpha-D-Manp-(1 leads to 3) ]-D-Manp and the trisaccharide alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-D-GlcNAc, all of which are 21-30 times more inhibitory than methyl alpha-D-mannopyranoside. The aromatic glycoside p-nitrophenyl alpha-D-mannopyranoside was also a strong inhibitor (30 times more inhibitory than methyl alpha-D-mannopyranoside), whereas the corresponding beta-D-glycoside was only a weak inhibitor (approximately as methyl alpha-D-mannopyranoside). A nearly identical pattern of inhibitory activity was observed with the fimbriae. This suggests that the combining site of the E. coli fimbrial lectin is in the form of an extended pocket on the surface of the lectin corresponding to the size of a trisaccharide and fitting best the structure alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-D-GlcNAc. Since p-nitrophenyl alpha-D-mannopyranoside is a strong inhibitor, the existence of a hydrophobic region in the combining site or close to it was assumed. The combining site of the Klebsiella pneumoniae fimbrial lectin is probably similar to that of E. coli, but that of the Salmonella typhimurium fimbrial lectin differs considerably. It appears that the combining sites of the three bacterial lectins tested exhibit preference for structures found in N-glycosylic oligomannoside units of mammalian cell surface glycoproteins.

Agglutination↗
PubMed1983

Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations.

Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.

Arachis↗
PubMed1983

Sugar-specific endocytosis of glycoproteins by Lewis lung carcinoma cells.

Lewis lung carcinoma cells from tumors, metastasis nodules, or from culture bind fluorescent derivatives of neoglycoproteins containing alpha-D-glucose residues: This binding is competitively inhibited by neoglycoproteins containing alpha-D-glucose, by mannan, and by several other neoglycoproteins. Cell binding and uptake of the fluorescent derivatives of the neoglycoproteins was quantified by lysing the cells with an alkylpolyol (MAC 19 or MAC 18) and measuring the fluorescence intensity of the supernatant. The amount of cell-associated neoglycoprotein was higher at 37 degrees C than at 4 degrees C with LLC from tumor. The binding and uptake were inhibited by glycoconjugates containing alpha-D-glucose. These results suggest the presence of sugar specific receptors in Lewis lung carcinoma cells which are involved in a sugar-specific binding and endocytosis phenomenon. The implication of the existence of a carbohydrate-binding protein on the surface of Lewis lung carcinoma cells are discussed with regard to the in vivo behaviour of these cells, especially in relation to their metastatic properties and to the possibility of using neoglycoproteins as specific carriers of cytotoxic drugs. Hybrid molecules of gelonin and neoglycoprotein containing alpha-D-glucose were used as targetted toxin: The targetted toxin was found to bind to and to enter the intact cells and was 100 times more toxic than free drug.

Animals↗
PubMed1983

Characterization of chicken lymphocyte subsets separated by peanut agglutinin.

The reactivity of chicken lymphocytes isolated from bursa, thymus, spleen, and peripheral blood (PBL) with peanut agglutinin (PNA) has been investigated. High numbers of cells binding PNA (PNA+ cells) were found in bursa (74.5%) and thymus (85.2%), as well as in the peripheral organs (spleen and PBL, 43.8 and 70%, respectively). In the latter organs, the levels of PNA+ cells exceeded by far the values reported for mammals. Separation of PNA+ and PNA- cells from different organs was achieved by selective agglutination with the lectin or by affinity chromatography on immobilized PNA. Experiments combining the use of limiting concentrations of neuraminidase to cleave off sialic acid from the surface membrane of splenocytes that do not bind PNA (PNA- cells), followed by agglutination with the lectin, suggested that the high binding of PNA to mature lymphocytes is most likely due to a low degree of sialylation of chicken cell surface glycoconjugates. The functional properties in vitro of PNA+ and PNA- spleen cell fractions were assayed in mitogen responses (PHA, Con A, PWM), the mixed-lymphocyte reaction (MLR), and the in vitro anti-SRBC antibody response. In all three systems the responses of the PNA+ fraction were found to be significantly higher than those of the PNA- cells. Mixing experiments revealed that irradiated (1500 R) PNA- cells preferably suppress pure T-cell responses of unfractionated spleen cells, whereas PNA- cells exerted a stronger suppressive effect on responses involving B cells. Thus, in the chicken, PNA may be a valuable tool to distinguish subsets of suppressor cells with different target specificity.

Animals↗
PubMed1983

Characterization of human umbilical cord blood lymphocyte subsets fractionated on immobilized peanut agglutinin.

Human cord blood mononuclear cells from single donors were separated on minicolumns of peanut agglutinin (PNA) coupled to Sepharose beads to yield two fractions: unbound cells (PNA-, 78%) that were eluted with phosphate buffered saline, and bound cells (PNA+, 22%) eluted with 0.2 M D-galactose. The total yield was 86% and the cells were fully viable. There was no enrichment for macrophages or for surface immunoglobulin positive (B) cells in either the PNA+ or PNA- subset. Only 26% of the PNA+ lymphocytes formed rosettes with sheep red blood cells, in contrast to 53% of the PNA- lymphocytes. The response of the PNA+ cells to mitogens and allogeneic stimulation was considerably lower than that of the PNA- cells, while that of the latter was higher than the response of the unseparated cells. The average ratios of response of the PNA+ to PNA- cells were 0.25 for PHA, 0.20 for concanavalin A, 0.15 for pokeweed mitogen, and 0.15 In the mixed lymphocyte reaction. when tested with monoclonal antibodies to lymphocyte surface markers, it was found that the PNA+ fraction was depleted of mature T cells and enriched in Ia positive cells. Our data show that the low reactivity of human cord blood mononuclear cells may be ascribed to the presence of a subpopulation of lymphocytes which are immunologically immature. They also provide further evidence that in humans the PNA receptor is a marker for immature T or B lymphocytes.

Cell Separation↗
PubMed1983

Specific lysis of antigenically irrelevant cells by cytotoxic T lymphocytes upon insertion of appropriate antigens into the target cell plasma membranes.

The importance of the membrane milieu to functional presentation of target cell (TC) antigens to cytotoxic T lymphocytes (CTL) was investigated by examining the interaction of CTL with TC plasma membrane (PM) fractions, in isolated form or integrated into antigenically irrelevant TC. Isolated ascitic vesicles, microsomes, and purified PM, containing serologically defined alloantigens that have been implicated as the relevant TC antigens, effectively, yet nonspecifically, inhibited the binding and lysis of TC by CTL. The same PM fractions, when inserted into antigenically irrelevant TC via vesicles containing Sendai virus components, rendered the TC susceptible to CTL-mediated lysis directed against the inserted antigens. These findings suggest that CTL interact specifically with TC determinants only when they are embedded in the proper membrane environment.

Animals↗
PubMed1983

Non-allergenic haemolysins in grass pollens and housedust mites.

25 batches of pollen (six common grasses, maize, short ragweed) and two batches of housedust mite (Dermatophagoides pteronyssinus) all contained haemolysins. The haemolysins of two grass pollens and of the housedust mites were of small MW (100-200 Da) and apparently non-allergenic. Both caused inflammation when injected into human skin, at doses that could be experienced naturally. The "allergic" airways diseases may not be entirely immunologically mediated.

Allergens↗
PubMed1983

Binding and phagocytosis of sialidase-treated rat erythrocytes by a mechanism independent of opsonins.

Rat peritoneal macrophages bind and phagocytoze homologous sialidase-treated erythrocytes at a rate which is dependent on the amount of sialic acid that has been removed from the cells. Increased binding of erythrocytes is observed after the removal of 10-20% of membrane sialic acid, while for phagocytosis at least 30-40% of this substance must be removed. With Vibrio cholerae sialidase only a partial (80%) hydrolysis of rat erythrocyte sialic acid is possible, whereas Arthrobacter ureafaciens sialidase leads to complete desialylation and therefore causes stronger binding and phagocytosis of the erythrocytes than the V. cholerae enzyme. Preincubation of peritoneal macrophages with sialidase impairs binding and phagocytosis. Experiments were performed to account for the stimulation of binding and phagocytosis observed in the presence of native, homologous serum. However, an involvement of immunoglobulins and complement factors of the classical and alternative pathway in the engulfment process has been excluded. Fibronectin, tuftsin and substance P have no influence, either. On the other hand, peanut agglutinin and Erythrina crystagalli agglutinin are potent stimulators of binding and phagocytosis of sialidase-treated erythrocytes, whereas soybean agglutinin has only little and limulin no influence at all. It is concluded that sialidase-treated erythrocytes, having been bound to the beta-galactose-specific lectin on the macrophage surface, are phagocytozed as a function of their number and binding strength to the macrophages. The influence of native serum and especially of the plant lectins on this process is discussed.

Animals↗
PubMed1982

Acquisition of mitogenic responsiveness by nonresponding lymphocytes upon insertion of appropriate membrane components.

The effect of insertion of plasma membrane components from lymphocytes responding to mitogens into the membranes of nonresponding cells using Sendai virus envelopes as vehicles was examined. T cells modified by B membranes were stimulated by lipopolysaccharide (LPS) to proliferate as well as to produce interleukin-2 activity. B cells modified by T membranes were stimulated by concanavalin A to proliferate and to produce interleukin-2 activity. B cells derived from C3H/HeJ LPS-nonresponder strain of mice, when modified by B membranes derived from the LPS-responder C3H/eb strain, acquired LPS responsiveness. These findings indicate that the inability of either T or B cells to respond to specific mitogens is due to the lack of suitable plasma membrane constituents and that by changing the membrane composition the lymphocytes can be endowed with new functions.

Animals↗