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I J Goldstein

Publications and source records attributed to I J Goldstein.

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

Purification and characterization of a new mannose-specific lectin from Sternbergia lutea bulbs.

A new mannose-binding lectin was isolated from Sternbergia lutea bulbs by affinity chromatography on an alpha(1-2)mannobiose-Synsorb column and purified further by gel filtration. This lectin (S. lutea agglutinin; SLA) appeared homogeneous by native-gel electrophoresis at pH 4.3, gel filtration chromatography on a Sephadex G-75 column, and SDS-polyacrylamide gel electrophoresis, These data indicate that SLA is a dimeric protein (20 kDa) composed of two identical subunits of 10 kDa which are linked by non-covalent interactions. The carbohydrate binding specificity of the lectin was investigated by quantitative precipitation and hapten inhibition assays. It is an alpha-D-mannose-specific lectin that interacts to form precipitates with various alpha-mannans, galactomannan and asialo-thyroglobulin, but not with alpha-glucans and thyroglobulin. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the SLA-asialothyroglobulin precipitation system, whereas D-glucose, D-galactose and L-arabinose were not. The lectin appears to be highly specific for terminal alpha(1-3)-mannooligosaccharides. The primary structure of SLA appears to be quite similar to that of the snow drop (Galanthus nivalis) bulb lectin which is a mannose-binding lectin from the same plant family Amaryllidaceae. The N-terminal 46 amino acid sequence SLA showed 76% homology with that of GNA.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of two different mannose-binding lectins from tulip bulbs.

Two lectins were isolated from the bulbs of Tulipa cv. Apeldoorn and their corresponding cDNA clones analyzed. The first, called TxLMII (second mannose-binding Tulipa hybrid lectin), is a novel mannose-binding tulip lectin. Based on its molecular structure, carbohydrate-binding specificity and amino acid sequence, TxLMII belongs to the superfamily of mannose-binding monocot lectins which are also found in representatives of the plant families Amaryllidaceae, Alliaceae, Orchidaceae and Araceae. Molecular cloning of the second lectin, called TxLCI (first Tulipa hybrid lectin with complex specificity), allowed determination unambiguously of the molecular structure of this previously described protein. In addition, evidence is presented that each TxLCI subunit possesses a mannose-binding site and an N-acetylgalactosamine-binding site, which act independently of each other. Both binding sites are located in a separate domain of the lectin polypeptide. Since the first domain of TxLCI shows sequence similarity to TxLMII, it is suggested that their genes evolved from a common ancestor.

Amino Acid Sequence↗
PubMed1996

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants.

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.

Animals↗
PubMed1996

Lectin binding characteristics of squamous cell carcinomas of the head and neck.

Lectins are polyvalent proteins of non-immune origin with exquisite carbohydrate binding specificity making them ideal for investigation of cell surface glycoprotein and glycolipid antigens. We examined the cell surface lectin binding phenotypes of 20 UM-SCC squamous cell carcinoma cell lines established from 17 patients with head and neck cancers using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of binding. Conconavalin A (Con A) from Canavalia ensiformis and the peanut agglutinin (PNA) from Arachis hypogaea bound all SCC cell lines tested and wheat germ agglutinin (WGA) from Triticum vulgaris bound to 12 of 13 tumor cell lines. The blood group O specific lectin UEA 1 from Ulex europeus also bound to all cell lines regardless of the donor blood type. Lectins of Dolichos biflorus (DBA) and Griffonia simplicifolia (GS I-B4 or BSA I-B4) with binding specificity for glycoproteins associated with blood group A and B respectively, had reactivity that did not directly correlate with blood group antigen expression. In contrast to the other lectins in our panel which exhibited broad reactivity with SCC antigens, the BSA-II lectin from Griffonia simplicfolia, (GS II or BSA II) which has sugar binding specificity for terminal non-reducing GlcNAc, did not bind to any of the screened cell lines. Our results demonstrate a common pattern of lectin-defined carbohydrate expression on the cell surface of squamous cell carcinomas of head and neck that appears promising in defining the malignant cellular phenotype. Lectin binding profile may be useful in differentiating benign from malignant histopathology.

Adult↗
PubMed1995

Site-directed mutagenesis studies on the lima bean lectin. Altered carbohydrate-binding specificities result from single amino acid substitutions.

The wild-type seed lima bean lectin (LBL), and recombinant LBL expressed in Escherichia coli show specificity for the human blood group A immunodominant trisaccharide GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-R. We have generated four site-specific mutants of LBL, two of which show altered specificity for extended carbohydrate structures. Four mutants, [C127Y]LBL, [H128P]LBL, [H128R]LBL and [W132F]LBL were expressed in E. coli. Two mutants show altered specificity for the substituent at the C2 hydroxy group of the penultimate Gal in the wild-type ligand which is alpha-L-fucose in the A trisaccharide. The mutant [C127Y]LBL showed specificity for the A disaccharide (GalNAc alpha 1-3Gal) and GalNAc alpha 1-4Gal, with free hydroxyl groups at the C2 position of Gal. The mutant [H128P]LBL bound the Forssman disaccharide structure GalNAc alpha 1-3GalNAc, in which the C2 hydroxyl group is substituted with an acetamido group. The third and fourth mutants, [H128R]LBL and [W132F]LBL, exhibited wild-type specificities, both recognizing the A trisaccharide. All of these mutant lectins bound the terminal GalNAc residues exposed on asialoovine submaxillary mucin, thus indicating that the monosaccharide-binding site had not been altered. We also determined that all but one mutant ([C127Y]LBL) retained the high-affinity binding site for N6 derivatives of adenine, indicative of tetramer formation; each mutant also expressed the low-affinity binding site for 8-anilinonaphthalene 1-sulfonate (1/monomer). Thus, by targeting two residues in LBL, we have identified a region of the protein that is part of the extended carbohydrate-binding site and which is specifically involved in the binding/recognition of substituents at the C2 position of the penultimate Gal of the A disaccharide. We have determined, by site-directed mutagenesis, that an essential Cys residue is involved in the specificity of LBL for the A trisaccharide.

Amino Acid Sequence↗
PubMed1995

Expression and function of beta 1 integrins on adherent and nonadherent Ehrlich ascites tumor cells.

Differences in integrin-mediated cell-matrix adhesion between two types of Ehrlich ascites tumor (EAT) cells, adherent and nonadherent EAT cells, have been studied. The adherent EAT (a-EAT) cells adhere to and spread on laminin- or fibronectin-coated plates, whereas the nonadherent EAT (na-EAT) cells do not. The adhesion of a-EAT cells to laminin or fibronectin requires the presence of both Ca2+ and Mg2+. Anti-human fibronectin receptor (alpha 5 beta 1 integrin) antiserum blocks a-EAT cells from adhering to both laminin- and fibronectin-coated plates. An RGD-containing peptide (GRGDSP) inhibits the adhesion of a-EAT cells to fibronectin-coated but not to laminin-coated plates. Taken together, these data suggest that a-EAT cells interact with laminin and fibronectin via different beta 1 integrins. As determined by immunofluorescence flow cytometry assay, both a-EAT and na-EAT cells express similar amounts of surface antigen(s) recognized by the same anti-alpha 5 beta 1 integrin polyclonal antibody that blocks a-EAT cells from adhering to laminin and fibronectin. The alpha 5 and beta 1 subunits expressed on both types of EAT cells appear to have the same molecular weight when analyzed by immunoblotting. The same amount of 125I-labeled membrane protein was isolated from both types of EAT cells on immobilized laminin and fibronectin affinity columns. These data indicate that na-EAT cells express similar amounts of cell surface integrins, and these integrins, when isolated, react with laminin and fibronectin in the same manner as the integrins of a-EAT cells, even though na-EAT cells are unable to adhere to laminin and fibronectin. Previous work has shown that both types of EAT cells express Neu5Ac alpha 2,3 Gal-beta 1, 4 GlcNAc residues on their cell surface and that they both react with Maackia amurensis lectin which specifically recognizes this carbohydrate structure. Here we show that both types of EAT cells attach to M. amurensis lectin-coated surfaces, but only a-EAT cells spread; this suggests that the differences between these two types of cells in responding to extra cellular matrix molecules may reside in some differences within the cell.

Animals↗
PubMed1995

Subcellular distribution of terminal alpha-D- and beta-D-galactosyl residues in Ehrlich tumour cells studied by lectin-gold techniques.

We have studied by high resolution in situ light and electron microscopic lectin-gold techniques the subcellular distribution of alpha-D-Gal residues using the Griffonia simplicifolia I-B4 isolectin and compared it with that of beta-D-Gal residues as detected with the Datura stramonium lectin in Ehrlich tumour cells grown as ascites or monolayer. The microvillar but not the smooth plasma membrane regions were labelled with the Griffonia simplicifolia I-B4 isolectin whereas both plasma membrane regions were equally well labelled with the Datura stramonium lectin. Elements of the endocytotic/lysosomal system such as coated membrane invaginations and vesicles, early and late endosomes and secondary lysosomes were positive for both alpha-D-Gal and beta-D-Gal residues. A particular feature of Ehrlich tumour cells is an elaborate tubular membrane system located in the pericentriolar region which is labelled throughout by both lectins and represents part of the endosomal system. In the Golgi apparatus labelling with both lectins was observed to commence in trans cisternae which is indirect evidence for a joint distribution of the sequentially acting beta 1,4 and alpha 1,3-galactosyl-transferases.

Animals↗
PubMed1995

Structure of mannose-specific snowdrop (Galanthus nivalis) lectin is representative of a new plant lectin family.

Tetrameric Galanthus nivalis agglutinin (50,000 M(r)) belongs to a super-family of alpha-D-mannose-specific plant bulb lectins known to be potent inhibitors of retroviruses. The 2.3 A crystal structure of this lectin complexed with methyl alpha-D-mannose reveals a novel three-fold symmetric beta-sheet polypeptide fold. Three antiparallel four-stranded beta-sheets, each with a conserved mannose-binding site, are arranged as a 12-stranded beta-barrel. The tetramer displays 222 symmetry. Pairs of monomers form stable dimers through C-terminal strand exchange. The so formed hybrid beta-sheets are the sites for high affinity mannose binding in the dimer interface. Occupancy observed at corresponding sites in other beta-sheets suggests a potential for twelve sites per tetramer.

Amino Acid Sequence↗
PubMed1994

Differential expression of cell surface sialoglycoconjugates on wild-type and cultured Ehrlich tumor cells as revealed by quantitative lectin-gold ultrastructural cytochemistry.

Three variants of the classical Ehrlich ascites tumor (EAT) cell have been studied by quantitative, sialic acid-specific, lectin-gold ultrastructural cytochemistry. Electron microscopic examination revealed pronounced differences in the surface morphology of the three cell variants. The wild-type Ehrlich cells (EAT-wt), grown in the peritoneal cavity of mice, exhibited a smooth surface profile. A variant form selected for growth as monolayer on basement membrane (EAT-c) showed a complex surface profile with numerous microvilli. The third variant (EAT-c/m), the cultured cells reinoculated into mice and passaged 20-25 times as ascites, presented a smooth surface profile similar to the EAT-wt cells. Quantitative single as well as double lectin-gold labeling revealed significant differences in the nature of cell surface sialoglycoproteins. The most significant finding was the presence of cell surface Neu5Ac alpha 2-6Gal residues as detected with the Sambucus nigra lectin on EAT-c and EAT-c/m cells, whereas EAT-wt cells contained little or none of such carbohydrate sequences. On the contrary, labeling by Maackia amurensis lectin, which recognizes the Neu5Ac alpha 2-3Gal beta 1-4GlcNAc sequence, was intense on all three Ehrlich cell variants; it was 20-60 times greater than alpha-2,6-linked sialic acid-containing glycoconjugates. Specific cell surface lectin binding combined with morphologic study appears to have identified a small subpopulation of cells within the ascites tumor that are capable of attaching to and growing on a basement membrane.

Animals↗
PubMed1994

Alpha-(2-->3)- and alpha-(2-->6)-sialyltransferase activities present in three variants of Ehrlich tumor cells: identification of the products derived from N-acetyllactosamine and beta-D-Gal-(1-->3)-alpha-D-GalNAc-(1-->O)-Bn.

We compared several sialytransferase activities related to synthesis of O-linked and N-linked sialyglycoproteins in Ehrlich ascites tumor cells that grow normally in murine ascites, but are not adherent nor grow in tissue culture (na-EAT cells), with those in cells that were selected to grow in tissue culture and adhere to extracellular matrices (a-EAT cells). Crude Golgi preparations from both cell types contained predominantly beta-D-Gal-(1-->3)-D-GalNAc alpha-(2-->3)-sialyltransferase activity. Sialylation of N-acetyllactosamine, lacto-N-tetraose, and benzyl alpha-D-GalNAc occurred at from 1 to 4% of that activity. Analysis, by ion-exchange HPLC at high pH, of sialylated N-acetyllactosamine showed that na-EAT cells sialylated beta-D-Gal-(1-->4)-D-GlcNAc mostly by alpha-(2-->3)-sialyltransferase, whereas beta-D-Gal-(1-->4)-D-GlcNAc alpha-(2-->6)-sialyltransferase activity was prominent in a-EAT cells. In addition, preparations from na-EAT cells formed significant quantities of an unknown tritiated product from CMP-[9-3H]sialic acid, suggesting at least one other difference in enzyme levels between the cell types. a-EAT cells reestablished in murine ascites for 11 passages retained the sialyltransferase levels characteristic of a-EAT cells. When viable cells were labeled with D-[3H]glucosamine, na-EAT cells formed larger amounts of sialic acid in O-linked glycoproteins than did a-EAT cells.

Amino Sugars↗
PubMed1994

The lectin Griffonia simplicifolia I-A4 (GS I-A4) specifically recognizes terminal alpha-linked N-acetylgalactosaminyl groups and is cytotoxic to the human colon cancer cell lines LS174t and SW1116.

The lectin GS I-A4 binds to terminal alpha-N-acetylgalactosaminyl (GalNAc) groups (which include the Tn antigen), but not to the closely related tumor-associated epitope, sialylated Tn antigen. The lectin also precipitates asialo OSM, but not its native sialylated form. Lectin histochemistry with human colonic tissues showed that GS I-A4 specifically stained specimens of colon cancer and colonic tissues from individuals with FAP; however, normal colonic tissues from patients without colonic disease were rarely stained with this lectin. Glycoconjugates bound by GS I-A4 were observed on the surface membranes of 2 human colon cancer cell lines, LS174t and SW1116, when fluorescein isothiocyanate (FITC)-conjugated GS I-A4 was used. GS I-A4 was toxic to these 2 human colon cancer cell lines in monolayer culture. A dose-response study conducted using 10-160 micrograms/ml, of GS I-A4 demonstrated significant dose-related toxicity against LS174t and SW1116 cells. At concentrations > 80 micrograms/ml, > 99% of LS174t and > 90% of SW1116 cells were killed. Four mM GalNAc specifically inhibited the cytotoxic effect of GS I-A4 (p < 0.001), whereas 4mM N-acetylglucosamine (GlcNAc) had no effect. Two other lectins that recognize terminal alpha-GalNAc residues, DBA and LBL, were significantly less cytotoxic to the colon cancer cells than GS I-A4. In the light of these findings, we speculate that GS I-A4 may have potential use as a diagnostic agent against colorectal cancer.

Acetylgalactosamine↗
PubMed1994

Isolation and characterization of an N-acetyl-D-galactosamine-binding lectin from Dutch Iris bulbs which recognizes the blood group A disaccharide (GalNAc alpha 1-3Gal).

A blood group type A disaccharide (GalNAc alpha 1-3Gal)-binding lectin has been purified from Dutch Iris (Iris x hollandica) bulbs to electrophoretic homogeneity by consecutive affinity chromatography on columns of immobilized asialofetuin-Sepharose 4B and Synsorb A disaccharide (GalNAc alpha 1-3Gal beta-O-(CH2)8CONH-Synsorb). This lectin agglutinates both native and trypsin-treated rabbit erythrocytes, but not human erythrocytes, irrespective of blood group type. Gel filtration chromatography on Sephacryl S-200 HR column and SDS-polyacrylamide gel electrophoresis revealed the lectin to be a heterodimer consisting of two peptide chains (27 and 34 kDa) linked by disulfide bonds. The carbohydrate binding specificity of the lectin was investigated by quantitative precipitation, hemagglutination inhibition, and precipitation inhibition assays. It is a Gal/GalNAc-specific lectin, with an extended carbohydrate combining site, which appears to be most complementary to GalNAc linked to the C-3 or C-6 hydroxyl group of galactose. As inhibitors, these disaccharides are approximately 30-60 times more potent than galactose and 4-8-fold more active than N-acetyl-D-galactosamine, whereas both the blood type A trisaccharide (GalNAc alpha 1-3[L-Fuc alpha 1-2]Gal) and the Forssman disaccharide (GalNAc alpha 1-3GalNAc) are noninhibitory, suggesting the importance of a free equatorial hydroxyl group at the C-2 position of the penultimate galactose for lectin binding; either an acetamido group or a fucosyl group at this position appears to cause steric hindrance, thus abolishing binding to the lectin.

ABO Blood-Group System↗
PubMed1994

The sequence of a second member of the lima bean lectin gene family and the expression and characterization of recombinant lectin in Escherichia coli.

The lima bean lectin recognizes terminal alpha-D-GalNAc groups and agglutinates human type A erythrocytes. We have cloned a portion of the gene encoding the alpha subunit of the lima bean lectin. The clone was obtained using the polymerase chain reaction and verified from a genomic clone encoding the mature protein of 253 amino acids. The deduced amino acid sequence has significant overall homology with other leguminous plant lectins and contains all of the known peptide sequences isolated from lima bean lectin (LBL). Southern blot analysis reveals the presence of several genes which hybridize to the cloned gene and which we propose are genes included in the lima bean lectin gene family. We report here the sequence, expression, and characterization of LBL 2, the second member of this gene family. Milligram quantities of soluble active recombinant lima bean lectin (rLBL) were obtained from Escherichia coli, using the T7 RNA polymerase expression system. SDS-polyacrylamide gel electrophoresis and Western blot analysis indicate expression of one protein band of about 27 kDa in induced E. coli cells. This protein cross-reacts with polyclonal antibodies raised against seed lectin (sLBL) and gave a reaction of identity with seed lectin by Ouchterlony double diffusion, specifically agglutinates type A blood cells, and is specifically inhibited by D-GalNAc. The isoelectric point of rLBL is 5.86, whereas those of the seed lectin subunits were determined to be 5.86, 5.58, and 5.20 (previously designated alpha, beta, alpha', respectively). rLBL binds to hydrophobic ligands independent of sugar binding, an observation similar to results obtained with sLBL. However, despite the similar activities described, several significant differences between recombinant and native lima bean lectin were found, including mobility on gel filtration, aggregation in solution, and its CD spectrum. These differences may be due to a number of factors, which will be discussed.

Amino Acid Sequence↗
PubMed1994

Tics and dyskinesias associated with stimulant treatment in attention-deficit hyperactivity disorder.

OBJECTIVE: To determine the incidence of tics or dyskinesias (T/D) and examine associated clinical factors in children treated with stimulant medications for attention-deficit hyperactivity disorder. DESIGN: Cross-sectional analysis of a clinic cohort with chart review. SETTING: Hospital-based clinical service within a division of developmental and behavioral pediatrics. PATIENTS: One hundred twenty-two children with attention-deficit hyperactivity disorder treated with stimulant medication. All children currently or recently treated were included. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Determinations were made of medication used, medication dosage, presence or absence of T/D, time of T/D onset, and history and family history of T/D. Incidence of T/D was 9.0% of children or 8.2% of medication trials. One child (0.8%) had development of Tourette's syndrome. Age, medication, dosage, history of tics, or family history of tics was not related to onset of T/D. CONCLUSION: Approximately 9% of children with attention-deficit hyperactivity disorder treated with stimulant medication had development of T/D, predominantly transient in nature, with less than 1% having development of chronic tics or Tourette's syndrome. Personal or family tic history, medication selection, or dosage was not related to onset of T/D.

Adolescent↗
PubMed1994

Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds.

A Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that the Mucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc alpha 1-3GalNAc)-related structures.

Amino Acid Sequence↗