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N Sharon

Publications and source records attributed to N Sharon.

At least 37 records · Page 2Linked to original sources
PubMed1997

Redefinition of the carbohydrate specificity of Erythrina corallodendron lectin based on solid-phase binding assays and molecular modeling of native and recombinant forms obtained by site-directed mutagenesis.

Binding of the N-acetyllactosamine-specific lectin from Erythrina corallodendron (ECorL) to four glycosphingolipids has been tested using the microtiter well assay. The role of several amino acids in the binding site region was studied by combining binding assays and molecular modeling for native and recombinant forms of the lectin. Seven single-point mutants at positions 106 (Y106A), 108 (Y108A, T), 218 (A218G), and 219 (Q219A, N or E) were investigated. A comparison with more than 30 known sequences of legume lectins showed that ECorL is unique in displaying a tyrosine residue or a structural equivalent at position 106. Analyses of the binding results obtained for mutants at positions 106 and 108 using molecular modeling point to complex conformational dependencies between these and several other residues around the binding site. Gln 219 was found to have a large conformational flexibility, which, paradoxically, favors the binding of N-acetyllactosamine-containing glycosphingolipids. Particularly significant is the fact that ECorL exhibits a higher affinity for Fuc alpha2Gal beta4GlcNAc beta-terminated glycosphingolipids than N-acetyllactosamine-terminated ones, in accordance with molecular modeling revealing a perfect fit of the alpha2-linked fucose in a cavity extending from the Gal beta4 binding pocket. These findings lead to a redefinition of the specificity of this lectin, where the affinity for the terminal Fuc alpha2Gal beta4GlcNAc beta trisaccharide should be considered in the first place. The possible biological significance of this specificity remains to be investigated.

Adsorption↗
PubMed1997

Blood group MN-dependent difference in degree of galactosylation of O-glycans of glycophorin A is restricted to the GalNAc residues located on amino acid residues 2-4 of the glycophorin polypeptide chain.

Glycophorin A (GPA) of human erythrocytes contains a minor number of unsubstituted GalNAc residues (Tn receptors) which are recognized by Moluccella laevis lectin (MLL). The lectin reacts better with blood group N- than M-type of GPA which suggests a higher number of Tn receptors in GPA-N than in GPA-M. To find out whether this difference is restricted to a defined domain of GPA, the N-terminal tryptic glycopeptides of GPA-M and GPA-N (a.a. residues 1-39) and their fragments obtained by degradation with CNBr (a.a. residues 1-8 and 9-39) were analyzed. The untreated and desialylated glycopeptides were tested as inhibitors of MLL in ELISA, and the content of GalNAc-ol was determined in the products of beta-elimination of the asialoglycopeptides by gas-liquid chromatography/mass spectrometry. The asialoglycopeptides 1-39 and 1-8 derived from GPA-N showed about 2 and 4 times higher content of non-galactosylated GalNAc residues, respectively, and higher reactivity with MLL than their counterparts derived from GPA-M, while asialoglycopeptides 9-39 of GPA-M and GPA-N did not show such differences. These results demonstrate that higher expression of non-galactosylated GalNAc in GPA-N than in GPA-M is confined to GalNAc residues located in the amino-terminal portion of GPA polypeptide chain, between the blood group M- and N-specific amino acid residues 1 and 5.

Acetylgalactosamine↗
PubMed1997

Central nervous system involvement at diagnosis in a case of pediatric CD30+ anaplastic large cell lymphoma.

Central nervous system (CNS) involvement in Ki-1/CD30 lymphoma is extremely rare, in contrast to the frequent involvement in other types of pediatric non-Hodgkin's lymphoma. No mechanism has yet been proposed to explain the sparing of the blood brain barrier in Ki-1/lymphoma. We present a 2-year-old boy who was admitted to the Department of Pediatric Hemato-Oncology due to lethargy, progressive breathing difficulties, massive diffuse lymphadenopathy, hepatosplenomegaly, and ichthyosis-like skin involvement with epidermolysis. A lymph node biopsy was compatible with Ki-1/CD30 anaplastic large cell lymphoma (ALCL). Bone marrow aspirate and biopsy demonstrated reactive hyperplasia. Cytogenetic analysis displayed hyperdiploid cells with 1p(-) in most cells. Cerebrospinal fluid examination showed pleocytosis with CD30+ cells. Possible mechanisms which could enable CNS involvement in this unusual case are discussed.

Central Nervous System Neoplasms↗
PubMed1997

Hodgkin's lymphoma in systemic onset juvenile rheumatoid arthritis after treatment with low dose methotrexate.

We describe the occurrence of malignant lymphoma as a possible complication of immunosuppression associated with low dose methotrexate (MTX) therapy for juvenile rheumatoid arthritis (JRA). A 6-year-old girl with systemic onset JRA who had received low dose MTX therapy for 16 months developed diffuse peripheral lymphadenopathy and enlargement of the lymph nodes in the mediastinum, hilum of the lungs, and liver. Lymph node histology disclosed mixed cellularity Hodgkin's lymphoma; the neoplastic cells were positive for CD30 and CD15, but negative for Epstein-Barr virus RNA or EBV latent membrane protein. After chemotherapy, the girl had complete remission of her disease lasting for 18 months; however, the disease relapsed and autologous peripheral stem cell transplantation was performed. Although the occurrence of lymphoma may be associated with autoimmune diseases, our observations suggest that in pediatric patients, the increasing use of low dose MTX therapy for JRA may be an additional factor for the development of lymphoproliferative disease.

Arthritis, Juvenile↗
PubMed1996

Mutational studies of the amino acid residues in the combining site of Erythrina corallodendron lectin.

High-resolution X-ray crystallography of the complex of the Gal/GalNAc-specific Erythrina corallodendron lectin with lactose identified the amino acid side chains that form contacts with the galactose moiety of the disaccharide. The contribution of these amino acids to the binding of different monosaccharides and oligosaccharides by the lectin was examined by site-directed mutagenesis. Replacement of Phe131, on which the galactose is stacked, by tyrosine, gave a mutant with the same hemagglutinating activity and carbohydrate specificity as the parent lectin, but replacement by alanine or valine resulted in loss of activity. Mutations of Ala88, Asp89, and Asn133 produced mutants that were also inactive whereas those of the other combining site residues, Tyr106, Ala218, and Gln219, were biologically active. None of the active mutants interacted with mannose or glucose. Thus, contrary to an earlier assumption. Ala218 is not responsible for the inability of E. corallodendron lectin to bind these sugars. Our findings also demonstrate that Gln219 is not involved in galactose binding in solution, even though this is implicated by the crystal data. Instead, our data suggest that Gln219 assists in the ligation of N-acetyllactosamine to the lectin, by interacting with the acetamide group of the disaccharide. Comparison with other legume lectins specific for mannose/glucose, galactose, N-acetylgalactosamine, L-fucose or N-acetylglucosamine, shows that only three of the combining site residues of E. corallodendron lectin occupy invariant positions both in their primary and tertiary structures. These residues are an aspartic acid and an asparagine corresponding to positions 89 and 133, respectively, in E. corallodendron lectin, and an aromatic residue, either phenylalanine (as Phe131 in this lectin), tyrosine or tryptophan. We therefore postulate that these three residues are essential for ligand binding by all such lectins, irrespective of their specificity.

Acetylgalactosamine↗
PubMed1996

Thermodynamics of monosaccharide and disaccharide binding to Erythrina corallodendron lectin.

Isothermal titration calorimetry measurements of the binding of 2'-fucosyllactose, lactose, N-acetyllactosamine, galactopyranose, 2-acetamido-2-deoxygalactopyranoside, methyl alpha-N-dansylgalactosaminide (Me-alpha-DNS-GalN), methyl alpha-D-galactopyranoside, methyl beta-D-galactopyranoside, and fucose to Erythrina corallodendron lectin (ECorL), a dimer with one binding site per subunit, were performed at 283-286 and 297-299 K. The site binding enthalpies, DeltaHb, with the exception of Me-alpha-DNS-GalN, are the same at both temperatures and range from -47.1 +/- 1.0 kJ mol-1 for N-acetyllactosamine to -4.4 +/- 0.3 kJ mol-1 for fucose, and the site binding constants range from 3.82 +/- 0.9 x 10(5)M-1 for Me-alpha-DNS-GalN at 283.2 K to 0.46 +/- 0.05 x 10(3) M-1 for fucose at 297.2 K. The binding reactions are mainly enthalpically driven except for fucose and exhibit enthalpy-entropy compensation. The binding enthalpies of the disaccharides are about twice the binding enthalpies of the monosaccharides in contrast to concanavalin A where the binding enthalpies do not double for the disaccharides. Differential scanning calorimetry measurements show that denaturation of the ECorL dimer results in dissociation into its monomer subunits. The binding constants from the increase in denaturation temperature of ECorL in the presence of saccharides are in agreement with values from isothermal titration calorimetry results. The thermal denaturation of ECorL occurs around 333 K, well below the 344-360 K denaturation temperature of other legume lectins of similar size and tertiary structure, undoubtedly due to the difference in its quaternary structure relative to other legume lectins. This is also apparent from the independent unfolding of its two domains.

Binding Sites↗
PubMed1996

Vincristine treatment triggering the expression of asymptomatic Charcot-Marie-Tooth disease.

A 16-year-old male suffering from Ewing's sarcoma of the pelvis was treated with vincristine as part of his chemotherapeutic protocol. The boy was never known to suffer from any neurological problems. His father had a mild limp, attributed to prolonged "taxi driving," that was never investigated medically. The first course of treatment, which included 2 mg of vincristine, resulted in clinical improvement. However, at the same time the patient developed severe weakness of both upper and lower limbs, areflexia, and gradually a pes cavus deformity. Nerve conduction studies were suggestive of severe peripheral sensorimotor neuropathy, axonal and demyelinative. A definite diagnosis of Charcot-Marie-Tooth was confirmed by molecular analysis showing the typical duplication of 1.5 megabases at 17 p11.2. This unique manifestation of vincristine neurotoxicity is reported and discussed.

Adolescent↗
PubMed1995

Two embryonal cancers after in vitro fertilization.

BACKGROUND: In vitro fertilization is not considered to be associated with an increased rate of pediatric malignancies, and only three have been reported in the literature. Two additional rare pediatric tumors in children conceived through this technique are reported. METHODS: Two children 12 and 18 months of age, developed hepatoblastoma and clear cell sarcoma of the kidney, respectively. They were both products of uneventful pregnancies induced by in vitro fertilization. No other environmental, prenatal, or family factor was found. RESULTS: The first child died after a failed remission induction with cisplatin, doxorubicin, and vincristine, whereas the second child is alive with no evidence of disease 18 months after diagnosis and treatment according to NWTS protocol. CONCLUSIONS: A possible association between in vitro fertilization and pediatric malignancies is suggested.

Antibiotics, Antineoplastic↗
PubMed1995

C-terminal post-translational proteolysis of plant lectins and their recombinant forms expressed in Escherichia coli. Characterization of "ragged ends" by mass spectrometry.

Electrospray mass spectrometry was used to accurately measure the molecular masses of single chain lectins from legume seeds and also of three recombinant lectins, expressed in Escherichia coli. The five single chain lectins, Erythrina corallodendron lectin, soybean and peanut agglutinins, Dolichos biflorus lectin, and Phaseolus vulgaris hemagglutinin E, all showed evidence of C-terminal proteolytic processing, in some cases to "ragged" ends, when their masses were compared to those expected from their cDNA sequences and their known carbohydrate chains. Recombinant forms of the lectins from E. corallodendron, soybean, and peanut also showed C-terminal trimming, but not to the same points as the natural forms. Discrepancies between the protein and cDNA sequences of the E. corallodendron lectin were resolved by combined liquid chromatography-mass spectrometry peptide mapping and protein sequencing experiments, and the presence of a second glycosylation site was demonstrated. Our data show that all of these lectins undergo C-terminal proteolytic processing of a readily attacked peptide segment. This trimming is frequently imprecise, and the resulting heterogeneity may be a major contributor to the appearance of isolectin forms of these proteins.

Amino Acid Sequence↗
PubMed1995

Nonopsonic phagocytosis of microorganisms.

Nonopsonic phagocytosis mediated by phagocyte receptors that recognize corresponding adhesins on microbial surfaces has attracted increasing interest as a potential host defense mechanism against extracellular pathogens and as a means of survival in the host for intracellular pathogens. Three types of nonopsonic phagocytosis involving carbohydrate-protein interactions (also termed lectinophagocytosis), protein-protein interactions, and hydrophobic interactions are discussed. A prominent receptor on phagocytic cells involved in recognizing pathogens belongs to the CD11/CD18 integrins. It mediates both opsonophagocytosis and nonopsonic phagocytosis and exhibits multiple specificity for different microbial adhesins. In other cases, similar specificity toward a microbial ligand (e.g. the Klebsiella pneumoniae capsule) is shared by dual molecules, one of which (e.g. the mannose-binding protein in serum) mediates opsonophagocytosis and the other (e.g. the macrophage mannose receptor) mediates nonopsonic phagocytosis of the microorganisms. In addition, we discuss how nonopsonic phagocytosis can trigger the phagocytes to release inflammatory agents and cause tissue injury. Further studies of the molecular mechanisms of nonopsonic phagocytosis, in particular those underlying the up-regulation of the phagocytic receptors by various agents, should lead to the development of new approaches for the prevention of infectious diseases.

Amino Acid Sequence↗
PubMed1995

Moluccella laevis lectin, a marker for cellular differentiation programs in mouse gut epithelium.

We have assembled a system for testing the hypothesis that changes in glycoconjugate production represent markers for defining developmental, spatial, and environmental influences on the proliferation and differentiation programs of various mouse gut epithelial cell lineages. Multilabel immunohistochemical methods were used to survey the interactions of purified lectins with 1) normal fetal, neonatal, and adult FVB/N mouse gut, 2) gastric and intestinal isografts harvested at various developmental stages, and 3) transgenic mouse models of intestinal epithelial cell hyperplasia, dysplasia, and/or neoplasia. As a demonstration of the system's utility, we used the recently purified, alpha-N-acetyl-D-galactosamine-specific, Moluccella laevis lectin (MLL). In the adult FVB/N mouse stomach, MLL only recognizes glycoconjugates produced by a population of nonproliferating neck and prezymogenic cells that occupy a pivotal point in the complex, migration-associated differentiation program of the zymogenic cell lineage. In the developing FVB/N stomach, MLL binds to members of the zymogenic and pit lineages even before morphogenesis of gastric units is completed. Expression of MLL epitopes in pit cells is restricted to the period before the gastric epithelium has completed its morphoregulatory program. Analysis of gastric isografts indicates that these lineage- and developmental stage-specific patterns of glycoconjugate accumulation are not influenced by normal luminal contents. In the adult FVB/N intestine, MLL binding can be used to operationally define variations in the differentiation programs of 1) members of the enteroendocrine and goblet cell lineages during their migration along the crypt-to-villus axis and 2) cells comprising the follicle-associated epithelium overlying Peyer's patches. Accumulation of MLL epitopes in villus-associated enterocytes does not appear to be affected when these cells are induced to reenter the cell cycle by simian virus 40 large T antigen (SV40 TAg). MLL reactivity is not diminished when enterocytes begin to dedifferentiate as a result of production of SV40 TAg, human K-rasVal12, and a dominant negative human p53 mutant. The lack of change in MLL binding properties may reflect the brief residence time of enterocytes on the villus. These results indicate that glycoconjugate production represents a very useful tool for studying gut epithelial cell biology. Preliminary studies suggest that this is also true in the human gut.

Animals↗