A lectin from Sesbania aculeata (Dhaincha) roots and its possible function.

A lectin was isolated from the roots of Sesbania aculeata. This is a glucose specific lectin having 39 kDa subunit molecular weight. The expression of this lectin was found to be developmentally regulated and observed to be the highest in the second week. The lectin was purified by affinity chromatography using Sephadex G-50 and found to have 28% homology with Arabidopsis thaliana lectin-like protein (accession No. CAA62665). The lectin binds with lipopolysaccharide isolated from different rhizobial strains indicating the plants interaction with multiple rhizobial species.

Anti-inflammatory effect of Ruta graveolens L. in murine macrophage cells.

Ruta graveolens L. (Rutaceae) is used for several therapeutic purposes worldwide. The present study is designed to investigate the effect of plant extract of Ruta graveolens on murine macrophage cells (J-774) challenged with lipopolysaccharide (LPS). LPS induces inflammatory response by stimulating the production of nitric oxide and other mediators. Significant inhibition (p=0.01 to p<0.002) of the LPS-induced nitric oxide production was observed in cells treated with plant extract in a concentration dependent manner. The inhibition observed for the extract was significantly higher than that observed for rutin, a flavonoid constituent of the plant. At 40 microM rutin, a comparable concentration of this flavonoid in the highest concentration (500 microg/ml) of plant extract was used in this study; a 20% inhibition (p=0.058) was observed. Inhibition in inducible nitric oxide synthase (inos) gene expression in the cells treated with the plant extract suggests an inhibition at the transcription level. Interestingly, a concomitant decrease in transcription of cyclooxygenase-2 (COX-2) gene has also been observed in cells treated with the plant extract and this inhibition is significantly higher than that observed with the highest concentration of rutin (80 microM) used in the study. As an inflammatory response, upregulation of nitric oxide synthase (iNOS) and COX-2 enzymes leads to production of pro-inflammatory mediators, namely nitric oxide and prostaglandins, respectively. Hence, the significant inhibitory effects on both of these inflammatory mediators unravel a novel anti-inflammatory action of this plant.

Interaction ofLabeo rohita lectin (LRP) with mycobacterial surface oligosaccharide.

A C-type mannose specific lectin (LRP) isolated from plasma of fresh water fishLabeo rohita showed 500 times higher specificity towards cell surface oligosaccharide (LAM) ofMycobacterium tuberculosis (H37Rv). Using biotinylated LRP, binding between lectin and LAM was demonstrated by ELISA and it was observed that even 3ng of oligosaccharides might be detected using only 1µg of biotinylated lectin.

Hemagglutinins in vegetative tissues of Bengal gram.

Hemagglutinating activity was identified in the roots and vegetative tissues of Bengal gram using rabbit erythrocytes. The activity in the roots appears to be similar to that of the seeds in respect to their sugar inhibition property. Seed and root extracts were found to have fetuin inhibitory agglutinating activity, whereas cotyledons showed different sugar specificity. However, 7-day cotyledons and roots from 3 and 7-day plants were also found to have galactose and N-acetyl-D-galactosamine inhibitory activity. Protein profile in different tissues during their developmental stages was analyzed by SDS-PAGE. A major band at 66 kDa was common to all tissues at different stages of their development except 3-day cotyledons where 2 major bands at 22 kDa and 74 kDa were observed. Two prominent bands at 74 kDa and 86 kDa were found along with 60 kDa protein in 3-day old roots whereas a major band at 33 kDa was found in 15-day old roots. Total protein content and specific activity of hemagglutinin were measured. Increase in specific activity with lower protein content was observed in cotyledons of 15-day old plant. Specific activity was eight times higher in the roots and four times more in leaf tissues of 7-day old plants in comparison to 15-day old plants. Stems also showed a significant increase of specific activity (p = 0.013) in 7-day old plants without any significant alteration in the protein content.

Expression, purification and characterization of peanut (Arachis hypogaea) agglutinin (PNA) from baculovirus infected insect cells.

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Galbeta1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

Interaction of peanut root lectin (PRA II) with rhizobial lipopolysaccharides.

Sugar specific binding of peanut root lectin (PRA II) to peanut specific bradyrhizobial lipopolysaccharides (LPS) was demonstrated by gel retardation assay and lectin based ELISA. Sephadex G-50 gel purified high molecular weight polysaccharides from NC 92 LPS bind PRA II most efficiently. Binding of NC 92 LPS only to PRA II and not to PNA, SBA and PSL by Western blot analysis suggests that this lectin-LPS interaction is tissue as well as species specific.

Partial characterization of the lipopolysaccharides of peanut specific Bradyrhizobium strains.

Hexoses, heptose, hexuronic acids, 2-keto-3-deoxyoctonic acid (Kdo) contents of the lipopolysaccharides (LPS) isolated from peanut (A. hypogaea) specific Bradyrhizobium strains NC 92, IGR 92, IGR 40 and IGR 6 were found to vary considerably. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed a heterogeneous LPS population and distinct heterogeneity was observed in NC and IGR strains. Sepharose gel filtration chromatography removed the hexose contaminants from NC 92 LPS. Polysaccharides obtained by mild acid hydrolysis of Sepharose purified NC 92 LPS were further purified on Sephadex G-50 gel filtration. Immunoblot analysis indicated cross reactivity amongst the peanut specific Bradyrhizohium strains but not with B. japonicum(SB 16). Antibodies raised against native NC 92 LPS showed higher binding towards the Sephadex purified polysaccharides suggesting the antigenic property of polysaccharide moiety in LPS molecule.

N-glycosylated polypeptides synthesized in the early phase of AcNPV infected insect cells.

Infected cell specific polypeptides (ICSP) in Autographa californica nuclear polyhedrosis virus (AcNPV) infected Spodoptera frugiperda cell line, Sf9, were radiolabelled with [14C] mannose and the labelled proteins were monitored at various time intervals. A major glycoprotein of molecular size of 64 kDa appears as early as 3h post infection (p.i.) which peaks at 12h p.i., but in presence of tunicamycin this protein is not labelled. Two other glycoproteins of molecular size 67 kDa and 63 kDa appear at 12h post infection. The glycosylation of these and eight other cellular proteins is also inhibited by tunicamycin. AcNPV propagated in tunicamycin treated Sf9 cells was found to cause delayed infectivity.

Purification of lectins from the stems of peanut plants.

The stem of the peanut plant contains two lectins, a methyl alpha-mannoside specific lectin (SL-I) and a lactose/cellobiose specific lectin (SL-II). These lectins are found to be developmentally regulated and maximum activities are observed in 3-4-weeks-old plants. The two lectins SL-I and SL-II have been purified from 3-week-old stem by affinity chromatography on Sephadex G-50 and guar gum matrices respectively. Both are glycosylated lectins and have the identical subunit molecular weight of 31 kDa.

Further characterization of glucose-specific peanut root lectin (PRA II).

Amino acid analysis of PRA II, a glucose-specific lectin isolated from 7 day-old peanut seedling roots shows that this lectin is rich in glycyl (103 per mole) and seryl residues (59 per mole), and poor in essential amino acids, the acidic amino acid content is higher than the basic amino acids and that its amino acid composition differs from its seed counterpart (PNA), although neither of the lectins contains cystein. PRA II has two carbohydrate binding sites per molecule as determined by equilibrium dialysis. Modifications of the specific amino acid residues of the lectin with group specific reagents indicate that hydroxyl group of tyrosine is involved in the binding of carbohydrate to PRA II.

Isolation and characterization of a lectin from peanut roots.

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephadex G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4-5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose.

A T-antigen-binding lectin from Channa leucopunctatus (Murrel) plasma.

The plasma of Channa leucopunctatus, which agglutinates human A,B,O blood-group erythrocytes nonspecifically, contains three separate agglutinating activities that are distinguishable by hemagglutination with specific blood-group erythrocytes. Blood group A agglutinating activity of the plasma was separated from the other two hemagglutinating activities by DEAE-cellulose column chromatography and further purified to homogeneity by affinity chromatography on 2-acetamido-2-deoxy-D-galactose coupled to epoxy-activated Sepharose 6B. The apparent homogeneity of the lectin was established by poly(acrylamide) gel electrophoresis, isoelectric focusing, immunodiffusion, and cross-immunoelectrophoresis. The native protein has a mol. wt. of 140,000 and two identical subunits. The isoelectric point of the affinity-purified lectin is 4.6. Amino acid analysis indicated high proportions of glycine, alanine, and aspartic acid. The lectin is a glycoprotein and it has a requirement for divalent cations, Ca2+ and Mg2+ or Mn2+, for hemagglutinating activity. 2-Acetamido-2-deoxy-D-galactose, 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside, T-antigenic disaccharide, and Forssman glycolipid are potent inhibitors.

Studies on hemagglutinins (lectins) from plasma of murrel fish, family Channidae.

Hemagglutinating activity can be identified in the plasma of different species of murrel fish. This activity may be divided into four types according to their agglutinability towards erythrocytes from different sources. Type I plasma agglutinates human blood group A erythrocytes, type II can agglutinate neuraminidase treated human A B O erythrocytes, type III shows no agglutinating activity towards human erythrocytes, while type IV agglutinates human erythrocytes non-specifically. All of them bind to DEAE-cellulose but elute out by different salt concentrations. Type IV plasma is found to be a combination of three separate hemagglutinins, which are separable by sequential binding to human A B O erythrocytes. Blood group A specific lectin activity is purified from this plasma using formalinised A group erythrocytes. The apparent homogeneity of this purified lectin is established by polyacrylamide gel electrophoresis, isoelectric focusing and immunodiffusion. This agglutinin is antigenically identical with that isolated from type I plasma by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose column. Their molecular weights are also found to be identical (Mr 140,000) in polyacrylamide gel electrophoresis, having two identical subunits. Forssman glycolipid (0.03 mM) was found to be the most potent inhibitor of agglutination, although Gal beta 1-3 GalNAc (0.09 mM) is also a good inhibitor. Exhaustive dialysis of the purified lectin (hemagglutinin) against EDTA denatures it irreversibly by dissociating it to its subunit structure. Thus human A group agglutinating activity isolated from type I and type IV plasma are identical.

Isolation and characterization of a new lectin from plasma of fish Channa punctatus.

A soluble lectin is purified to apparent homogeneity from plasma of Channa punctatus by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose. The lectin has 140 kDa native molecular mass and 68 kDa subunit molecular mass, as determined by native and sodium dodecyl sulphate denaturing polyacrylamide gel electrophoresis, respectively. The lectin agglutinates human A and AB blood groups and rat, mice and guinea pig erythrocytes in the presence of Ca2+ and Mg2+ or Mn2+ ions. These divalent cations, but not thiol group, are obligatory requirements for the lectin activity. Gal(beta 1----3)GalNAc (0.09 mM) is the most potent inhibitor of the lectin.

Separation and concentration of some platinum metal ions with a new chelating resin containing thiosemicarbazide as functional group.

A new chelating ion-exchange resin containing thiosemicarbazide as functional group and based on macroreticular polystyrene-divinylbenzene (8%) has been prepared. Its sorption characteristics for palladium(II), platinum(IV), rhodium(III), ruthenium(III) and iridium(III) have been studied. These platinum metal ions can be quantitatively separated by sorption on this chelating resin and selective elution. The resin is highly stable in acid and alkaline solution.

The uncoupling of macromolecular synthesis from cell division in SV3T3 cells by glucocorticoids: the imposition of a G2 block.

Through a receptor-mediated process glucocorticosteroids block cell division by 20-45 hours in SV40-transformed 3T3 (SV3T3) mouse fibroblasts growing in a low calf serum (0.30% v/v) medium containing biotin. However, the rate of DNA synthesis, determined at various times after dexamethasone addition by the incorporation of radioactive thymidine into acid-insoluble material, is not inhibited by this steroid as late as 66 hours. A modest decrease is observable by 91 hours. There is also no reduction in the uptake of exogenous thymidine into acid-soluble cellular pools. Similarly, RNA synthesis and the uptake of radioactive uridine are not affected by the glucocorticoid up to 69 hours. Measurements of the amounts of cellular DNA (by the fluorescent dye, 4', 6-diamidino-2-phenylindole) and protein revealed that both macromolecules are present in elevated quantities in steroid-treated cells. (The constancy of the protein content in the nonproliferative stage suggests that protein synthesis and degradation are occurring at equal rates.) If the steroid is removed and fresh 10% calf serum medium added, cell division commences (even if nearly 90% of protein synthesis is inhibited by cycloheximide) as early as 45 minutes later such that by 2 hours the viable cell count increases by as much as 70%. Since the growth curve after recovery resembles a step function, it appears that the cells are partially synchronized by the glucocorticoid. These results demonstrate that the glucocorticoid cytostatic effect in SV3T3 cells is the result of a block not in G1, as previously thought, but in G2.

Physicochemical and morphological characterization of a new allotransplantable mammary carcinoma ascites subline, TA3-St/ticol/-A.

The TA3-St/ticol ascites cell (I), immunoselected from the strain-specific TA3-St mammary carcinoma ascites cell of the strain A mouse for decreased H-2a antibody-binding capacity, underwent a spontaneous transition in vivo to a new cell line, TA3-St/ticol/-A (II). Line II was more allotransplantable than was the parental line (line I), and its absorptive capacity for anti-H-2a antibody was manyfold less than line I. Disruption of line II cells by lyophilization did not increase the absorptive capacity, in contrast to its marked enhancement in the TA3-Ha ascites cell under similar conditions. An explanation for the enhanced allotransplantability of line II may be related to either a loss of H-2a antigens or altered macromolecular structures at the cell surface: sialic acid, consisting of 93% N-glycolylneuraminic acid for line II and 9% for line I; altered chemical structures of cell surface glycoproteins, particularly a high-molecular-weight glycoprotein present in much greater proportion in line II than in line I; a macromolecular complex released by a protease from line I, but not line II; and I being agglutinable by concanavalin A, but not line II. Electron microscopy showed line II to be more pleomorphic and less rounded than was line I. Under high-resolution electron microscopy, the cell surfaces of both allotransplantable cells, lines II and I, exhibited thin filamentous material, material not observed at the surface of the nonallotransplantable TA3-St ascites cell.

Cell surface characteristics of an allotransplantable TA3 ascites subline resulting from a process of immunoselection.

Comparison of the cell surface characteristics of the parental strain-specific TA3-St ascites cell (I) of the strain A mouse and the more allotransplantable TA3-St/ticol ascites cell (II), immunoselected for reduced absorption of anti-H-2a antibody from Cell I, revealed the following. Cell II, like Cell I, possessed no detectable epiglycanin at its surface, as neither cell absorbed more than 0.5% as much of the antiepiglycanin antibody as was absorbed by the epiglycanin-containing allotransplantable TA3-Ha ascites cell. Tritium-labeled glycoproteins, with polyacrylamide slab gel electrophoresis with sodium dodecyl sulfate and with isoelectric focusing of detergent-treated cells, exhibited marked quantitative differences, but qualitative differences were not established. Glycopeptides cleaved from each cell by proteolysis and fractionated by gel filtration gave similar elution profiles, and the column fractions possessed similar carbohydrate and amino acid compositions. Less sialic acid (170 micrograms/10(9) cells) was removed by neuraminidase from Cell II than from Cell I (270 micrograms/10(9) cells), and the compositions (9% N-glycolylneuraminic acid for Cell II and 20% for Cell I) were different. Transmission and scanning electron microscopy showed rough irregular folds and ridges on the surfaces of each cell, but Cell II appeared more pleomorphic and less rounded than did Cell I. High-resolution transmission electron microscopy showed filamentous material at the surface of Cell II, but not at the surface of Cell I.

Studies on the possible chemical, immunochemical and morphological differences at the cell surfaces of immunosensitive and immunoresistant, Moloney virus-induced, lymphoma cell-lines.

Comparison was made of several cell-surface parameters in the immunosensitive, Moloney virus-induced, mouse lymphoma, YAC, and its immunoresistant variant, YACIR. The characteristics of the two cell lines appeared to be similar by most of the criteria employed. The poly(acrylamide)-gel electrophoresis (with sodium dodecyl sulfate) patterns, after staining with Coomassie Brilliant Blue, of detergent-solubilized materials, appeared to be identical. After elution from a gel filtration column, no major differences were observed in the protein profiles of material cleaved from viable cells by proteolysis. Scanning and transmission electron microscopy revealed no major differences between the YAC and YACIR cells. The concentration of the lectins, Ricinus communis agglutinin, concanavalin A, wheat-germ agglutinin, and Solanum tuberosum (potato) agglutinin, required to agglutinate viable cells of the two lines were not significantly different. Neither cell was agglutinated by the lectins from Dolichos biflorus or Vicia graminea. Significant differences were, however, observed in the concentrations of lectin from Arachis hypogaea (peanut) needed to agglutinate the two cells. Although similar amounts (184-188 micrograms/10(9) cells) of sialic acid were released from viable cells by neuraminidase (V. cholerae), striking differences were observed in the composition of this material: 48% of N-glycolylneuraminic acid for YAC and 15% for YACIR. The remainder was N-acetylneuraminic acid for each cell line.

Preparation and properties of a new chelating resin containing 2-nitroso-1-naphthol.

A new chelating ion-exchange resin based on a macroreticular polystyrene-divinyl benzene copolymer and containing 2-nitroso-1-naphthol as the functional species has been synthesized. It is highly stable in acidic and alkaline solutions. Its sorption characteristics for V(V), Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Hg(II), Pd(II) and U(VI) have been investigated over the pH range 1.0-7.0 and the exchange-capacities have been found to be generally higher than those of a similar resin containing 1-nitroso-2-naphthol.