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N Sharon

Publications and source records attributed to N Sharon.

At least 109 records · Page 6Linked to original sources
PubMed1986

Lectins as probes of insulin receptor carbohydrate composition: studies in glycosylation mutants of Chinese hamster ovarian cells with altered insulin binding.

We recently reported marked differences in insulin binding properties in Chinese hamster ovarian cell mutants with genetic defects in protein N-glycosylation. To further characterize the role of insulin receptor carbohydrates, we have now studied the effect of lectins on [125I]insulin binding to wild type (WT) Chinese hamster ovarian cells and to two mutant cell lines: B4-2-1, to which insulin was previously shown to bind with higher affinity than normal, and Lec 1, to which insulin binds with much lower affinity. The results show that of four lectins that bound to WT cells; only wheat germ agglutinin and phytohemagglutinin-E competed with insulin binding to these cells, while Concanavalin A (ConA) and Erythrina cristagalli agglutinin (ECA) did not. After solubilization of the cells, however, a potent inhibition of insulin binding was also seen with ConA and ECA. This suggests that sugar determinants for ConA and ECA are present on the insulin receptor, but are not accessible at the surface of the cells. Mutant B4-2-1 cells, which are deficient in mannosylphosphoryldolichol synthase and beta-galactosidase, differed from WT cells in that ECA and ConA potently inhibited insulin binding in intact cells. This suggests that these lectin binding sites of or near the insulin receptor are more accessible at the cell surface in this mutant cell line. Mutant Lec 1 cells, deficient in N-acetylglucosaminyl-transferase I, cannot process N-linked carbohydrates from their oligomannose to their complex forms. In these cells, marked differences in the pattern of lectin inhibition were observed compared to that in WT or B4-2-1 cells. ConA exerted a strong inhibition of insulin binding to solubilized cell preparations. Its effect on intact cells was modest however, suggesting that in this mutant line exposure of the insulin receptor at the cell surface is not different from that in the WT cells. Neither ECA nor PHA inhibited [125I]insulin binding to either intact or solubilized Lec 1 cells, suggesting that the absence of sugar determinants for these two lectins may play a role in the very low insulin binding affinity previously reported in this cell line. In conclusion, these indirect studies with lectins suggest that the carbohydrate units of the insulin receptor are heterogeneous. While some may be important for proper exposure of the receptor at the cell surface, others may play a role in more intrinsic receptor properties.

Animals↗
PubMed1985

Binding of [1-13C]galactose-labeled N-acetyllactosamine to Erythrina cristagalli agglutinin as studied by 13C-NMR.

The equilibrium binding kinetics of enzymatically prepared N-acetyllactosamine to the lectin from Erythrina cristagalli have been investigated by 13C-NMR spectroscopy. Under the experimental conditions used, NMR signals in the spectrum, corresponding to both the free and bound disaccharide species, were observed for the first time. This has permitted the simultaneous determinations of the equilibrium binding constant and the number of binding sites per lectin molecule. At the relatively high lectin concentrations used (0.3-0.87 mM), the association constants determined at 31 degrees C (approximately 6 X 10(3) M-1) are typically lower then those obtained by other methods employing much lower lectin concentrations. Extrapolation of the experimentally observed values to infinite dilution gave a better fit of the data (Ka approximately 1.4 X 10(4) M-1) with the binding constant determined by other methods (K approximately 1.1 X 10(4) M-1). The sugar residence time on the lectin (approximately 0.2 s) was determined directly from the signal's line-width using total line-shape analysis. Similar NMR experiments may permit an analysis of the interaction of the lectin with glycoproteins and cells labelled with 13C-enriched galactose residues. Moreover, information on lectin-galactose interactions at the binding site may be obtained by using galactose labeled at various carbons.

Amino Sugars↗
PubMed1985

Detection and enumeration of monocytes in human blood with peanut agglutinin.

Binding of peanut agglutinin (PNA) to normal human peripheral blood mononuclear cells was analyzed on a cell sorter, and compared to the binding of the monocyte specific monoclonal antibodies Mac-1 and Leu-M3. Each of the reagents labeled 9-11% of the mononuclear cells and similar binding patterns were observed. Of the PNA+ cells, 67% adhered to plastic petri dishes, whereas 76% of Mac-1+ cells were adherent. No competition for binding was observed between PNA and Mac-1 on the one hand, or PNA and Leu-M3 on the other. In double staining experiments, about 10% of the cells, comprising 80% of the monocytes, were PNA+ Leu-M3+. Our results show that PNA can serve for the identification and enumeration of monocytes in human peripheral blood.

Antibodies, Monoclonal↗
PubMed1985

Binding of N-dansylgalactosamine to the lectin from Erythrina cristagalli as followed by stopped-flow and pressure-jump relaxation kinetics.

The binding kinetics of N-dansylgalactosamine to the lectin from Erythrina cristagalli have been studied using stopped-flow and pressure-jump chemical relaxation by monitoring ligand fluorescence. Both methods gave results which are consistent with a simple bimolecular association reaction. The association rate constant, k + 1 = 4.8 X 10(4) M-1 s-1, is far too low to be controlled by diffusion; the dissociation rate is 0.4-0.66 s-1, depending upon the method of determination and the experimental conditions. Identical reaction-rate parameters were obtained at pH 7.3, where soluble aggregates can be present in the lectin solution and at pH 4.7 where such aggregates are absent. The slow rates of carbohydrate binding seem to be characteristic for most lectins and lend support to the idea that they are evolutionary related and have structurally similar binding sites. Analysis of the relaxation amplitudes of the pressure-jump experiments yielded a molar reaction volume change, delta V0, upon binding of +7 ml/mol. This volume change can be caused by desolvation of the ligand upon binding.

Binding Sites↗
PubMed1985

Role of fibronectin in attachment of Streptococcus pyogenes and Escherichia coli to human cell lines and isolated oral epithelial cells.

We studied the binding of cells of Streptococcus pyogenes and mannose-sensitive Escherichia coli to human fibroblast cell lines and isolated buccal epithelial cells in relation to the cell-associated endogenous or exogenous fibronectin of the host cells. The degree of bacterial binding to cell lines correlated directly with the content of endogenous fibronectin on the surface of the cultured cells, although the correlation was better with S. pyogenes than with E. coli. The addition of exogenous plasma fibronectin to the cell lines or oral epithelial cells enhanced binding of S. pyogenes but suppressed binding of mannose-sensitive E. coli. These findings are consistent with the notion that exogenously acquired fibronectin on the surface of host cells modulates bacterial adherence by providing attachment sites for certain pathogens, such as S. pyogenes, and by blocking receptors for others, such as mannose-sensitive E. coli.

Adhesiveness↗
PubMed1984

Binding of simple carbohydrates and some of their chromophoric derivatives to soybean agglutinin as followed by titrimetric procedures and stopped flow kinetics.

The number of carbohydrate-binding sites of the GalNAc-specific lectin is four per tetramer. The binding parameters of N-acetyl-D-galactosamine and methyl-N-acetyl-alpha-D- galactosaminide , were determined by titrating the perturbation in the absorption spectrum of the protein. For D-galactosides, it was necessary to use p-nitrophenyl-N-acetyl-beta-D- galactosaminide as an indicator in substitution titrations. The association constants K were determined at several temperatures yielding 2.4 X 10(4) M-1 at 25 degrees C with delta H degree' = -45 kJ mol-1 and delta S degree' = -67 J X K-1 mol-1 for methyl-N-acetyl-alpha-D- galactosaminide and 1.0 X 10(3) M-1 at 25 degrees C, delta H degree' = -38 kJ mol-1 and delta S degree' = -69 J X K-1 mol-1 for methyl-alpha-D-galactoside. The increase in K by a factor of 25 caused by the acetamido group is largely enthalpic . Whenever different methods were used to determine the association constant of a given compound, the agreement was excellent. The observed changes in absorption or fluorescence of all chromophoric carbohydrate derivatives used are specific for the binding of carbohydrates. For large aromatic beta- aglycons such as p-nitrophenyl or 4-methylumbelliferyl groups, the increase in K of the N-acetyl-D- galactosaminide moiety is by a factor of 2 or less, but for a large N-5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group this factor is about 20 as compared with the acetyl group. The concomitant 10-fold increase in dansyl fluorescence, also observed with four other GalNAc-binding lectins together with a favorable and large delta S degree' = +60 J X K-1 mol-1 strongly point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The results of stopped flow kinetics with 4-methylumbelliferyl-N-acetyl-beta-D- galactosaminide and the lectin are consistent with a simple mechanism for which k+ = 1.1 X 10(4) M-1 S-1 and k- = 0.4 S-1 at 25 degrees C. This k- is slower than for any monosaccharide-lectin complex reported so far.

Acetylgalactosamine↗
PubMed1984

Chemiluminescence and lymphocyte proliferation: parallelism in collaboration between subpopulations of thymus cells for both types of responses.

Rat thymocytes respond to exposure to phytomitogens by oxidant generation as detected by chemiluminescence in presence of luminol. Maximal chemiluminescent response to a wide variety of plant lectins was obtained only after the cells had been incubated for 18 hours at 37 degrees C but not at 4 degrees C. This temperature dependence and the necessity for intact protein-and RNA-synthetic machinery during the incubation period indicate the occurrence of differentiation of thymocytes as they develop the capacity for chemiluminescent response. Furthermore, adherent-phagocytic cells play an essential collaborative role during this differentiation. A remarkable parallelism was shown to exist in the capacity of a cell subset to respond to concanavalin A by DNA synthesis and the ability of the same subset to respond by chemiluminescence. The latest-sedimenting small lymphocytes after velocity sedimentation of thymus cells develop the capacity for DNA-synthetic as well as chemiluminescent responses to concanavalin A only if allowed to collaborate with a population of early-sedimenting adherent-phagocytic cells.

Animals↗
PubMed1984

Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand.

Erythrina cristagalli agglutinin, a dimeric lectin [J.L. Iglesias, et al. (1982) Eur. J. Biochem. 123, 247-252] was shown by equilibrium dialysis to be bivalent for 4-methylumbelliferyl-beta-D-galactoside. Upon binding to the lectin, this ligand showed a difference absorption spectrum with two maxima (at 322 and 336 nm) of equal intensity (delta epsilon = 1.2 X 10(3) M-1 cm-1). A similar spectrum with a comparable value of delta epsilon was obtained with 4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide. Binding of methyl-alpha-D-galactoside, lactose, and N-acetyllactosamine all produced small but equally intense protein difference spectra with a maximum (delta epsilon = 2.8 X 10(2) M-1 cm-1) at 291.6 nm. Upon binding of N-dansyl-D-galactosamine to the lectin, there was a fivefold increase in fluorescence intensity of this ligand. The association constant for N-dansyl-D-galactosamine was caused by a very favorable delta S degree of the dansyl group without affecting the strictly carbohydrate-specific character of binding. N-Dansyl-D-galactosamine was employed as a fluorescent indicator ligand in substitution titrations. This involved the use of simple carbohydrates, N-acetyllactosamine, and oligosaccharides which occur in the carbohydrate units of N-glycoproteins; the latter were Gal(beta 1----4)GlcNAc(beta 1----2)Man, Gal(beta 1----4)GlcNAc(beta 1----6)Man, and Gal(beta 1----4)GlcNAc(beta 1----6)[Gal(beta 1----4)GlcNAc(beta 1----2)]Man. The titrations were performed at two temperatures to determine the thermodynamic parameters. In the series N-acetyl-D-galactosamine, methyl-alpha-D-galactoside, and lactose, -delta H degrees increased from 24 to 41 kJ mol-1; it increased further for N-acetyllactosamine and then remained unchanged for the N-acetyllactosamine-containing oligosaccharides (55 +/- 1 kJ mol-1. This indicated that the site specifically accommodated the disaccharide structure with an important contribution of the 2-acetamido group in the penultimate sugar. Beyond this, no additional contacts seemed to be formed. This conclusion also followed from considerations of delta S degrees values which became more unfavorable in the above series (-23 to -101 +/- 4 J mol-1 K-1); the most negative value of delta S degrees was observed with N-acetyllactosamine and the three N-acetyllactosamine-containing oligosaccharides.

Amino Sugars↗
PubMed1984

Nature of the receptor sites for galactosyl-specific lectins on human lymphocytes.

The nature of the receptors for four lectins specific for D-galactosyl residues was examined in human lymphocytes. The cells were fixed with formaldehyde to avoid subsequent cell lysis, treated with pronase, sialidase and organic solvents, and the binding of the lectins to the treated cells measured. The results show that the bulk of the receptors for peanut agglutinin (PNA) and ricin (RCA 60) are glycoproteins, whereas those for Ricinus communis agglutinin (RCA 120) and soybean agglutinin (SBA) are distributed nearly equally between membrane glycoproteins and glycolipids.

Glycolipids↗
PubMed1984

Immunofluorescent evidence of prior herpes simplex virus type-2 infection in prostate carcinoma.

The finding of herpes simplex virus type-2 (HSV-2) particles in prostatic carcinoma (PCa) tissue has led to speculation that the virus might cause this disease. We studied 27 PCa and 33 benign prostatic hyperplasia (BPH) specimens for the presence of HSV-2 antigens by indirect immunofluorescent staining to HSV-2 using commercially prepared rabbit anti-HSV-2 and fluorescein-tagged goat and anti-rabbit antibody. The slides were randomly number coded by an impartial referee then read independently by each investigator. In cases of disagreement, new slides were prepared and read until agreement. The code was then broken. Seven of 27 PCa specimens and 8 of 33 BPH specimens showed positive staining. By contingency table analysis, the results were not statistically different (chi 2 = 0.0224; p greater than 0.8). In our series, there is no difference in the prevalence of HSV-2 staining between PCa and BPH. Further examination of our data failed to show any difference in the prevalence of staining for HSV-2 based on whether the source of the tissue was surgical or autopsy. We conclude that HSV-2 infection of the prostate is common (15/60 = 25%) but probably has no causal relationship to PCa.

Antigens, Viral↗
PubMed1984

Adenosine deaminase activity in lymphocyte subpopulations of B-16 melanoma and normal C57BL bearing mice.

The activity of adenosine deaminase (ADA) was measured in thymus and spleen subpopulations separated by peanut agglutinin (PNA) of melanoma B-16 C57BL bearing mice and normal age-matched C57BL mice. Groups of 10 mice were used each time and the experiments were repeated 6 times. The adenosine deaminase activity in the PNA+ thymocytes of B-16 bearing mice was about 2.5 times lower than that of the normal C57BL mice while the ADA activity in the PNA+ fraction of spleen of the B-16 melanoma bearing mice was 2.5 times higher. These results demonstrate that the tumor burden probably induces a different redistribution and traffic of lymphocytes from one lymphopoietic organ to another. This traffic can also explain the thymus involution and spleen enlargement found in the B-16 mice.

Adenosine Deaminase↗
PubMed1984

Surface galactolipids of wheat protoplasts as receptors for soybean agglutinin and their possible relevance to host-parasite interaction.

Soybean agglutinin, a lectin specific for N-acetyl-d-galactosamine and d-galactose, was previously shown to agglutinate wheat leaf protoplasts (Larkin 1978 Plant Physiol 61: 626-629). We investigated the receptors for soybean agglutinin on the plasma membrane of these protoplasts. After treatment of the protoplasts with galactose oxidase, they were no longer agglutinated by the lectin, whereas upon reduction of the galactose oxidase-treated protoplasts with sodium borohydride the susceptibility to agglutination was restored. Analysis of the glycolipids of protoplasts surface labeled by the galactose oxidase-borotritide method, revealed that the radioactivity was mainly present in monogalactosyldiglyceride and digalactosyldiglyceride. The same galactolipids were identified as the only receptors for soybean agglutinin by direct binding of the (125)I-labeled lectin to a thin layer chromatogram of the glycolipids of wheat leaf protoplasts.

Journal Article↗