Characterization of a yeast D-mannan with an alpha-D-glucosyl phosphate residue as an important immunochemical determinant

Conditions for the reaction of concanavalin A and dextranase with glutaraldehyde have been established to give a soluble, intermolecularly cross-linked conjugate possessing both dextranase and concanavalin activities. Evidence is presented that the dextranase and concanavalin molecules are linked to each other in the conjugate. The conjugate gives a different pattern of hydrolysis products on incubation with dextran than does dextranase.     

Protein-protein conjugation on a lectin matrix.

An efficient method of protein-protein conjugation yielding primarily monoconjugates is described. A glycoprotein enzyme, invertase, was ‘spaced-out’ on a succinyl concanavalin A sepharose matrix and reacted with 1% glutaraldehyde. The excess glutaraldehyde was washed out and a second, non-glycoprotein, enzyme, urease, was reacted with the ‘activated’ invertase. The column was washed till the washings were free of enzymatic activity. On elution with α-methyl glucoside both enzymes were detected in the eluate. Resolution on Sepharose 6B revealed that the eluted invertase was completely conjugated to urease. The molecular size of the conjugate suggested that it was a monoconjugate. The glutaraldehyde treated enzyme retained its immunological reactivity in the conjugate. This method of protein-protein conjugation is applicable if one of the two involved proteins is a glycoprotein.     

Photosensitization by chlorine and porphyrin causing structural damage of erythrocytes

Kinetics of hemolysis of human erythrocytes photosensitized by porphyrin and chlorin derivatives was investigated by small angle light scattering method. The compounds used were arranged in the following order of photosensitizing activity decrease: ethylendiamide of chlorine e6, chlorine e6 dimethylester chlorine e6 haematoporphyrin diacetate tetracarboxyphenylporphyn. The substances having a greater number of binding sites with liposomes (chlorine e6 dimethylester) or with albumin (ethylendiamide of chlorine e6) showed the greatest photohemolytic activity. The photohemolytic activity of porphyrins was suggested to depend on the number of the pigment molecules bound by erythrocytes membranes.     

Entry of diphtheria toxin linked to concanavalin A into primate and murine cells

Diphtheria toxin linked by a disulfide bridge to concanavalin A was highly toxic to HeLa S3 and Vero cells, as well as to murine L cells. The cells could be protected with alpha-methyl mannoside, indicating that the conjugate binds mainly through its concanavalin A moiety. Treatment of Vero cells with phospholipase C, TPA (12-O-tetradecanoylphorbol-13-acetate), and vanadate, which strongly reduce the ability of the cells to bind free diphtheria toxin, had little protective effect against the conjugate, whereas SITS (L-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid), which inhibits diphtheria toxin binding, as well as the subsequent entry, protected Vero cells, but not L cells. Both types of cells are protected against the conjugate by NH4Cl and monensin, indicating that an acidified compartment is necessary for entry into the cytosol. Exposure of cells, bound with surface conjugate, to low pH induced entry of the toxin into Vero cells, but not into L Cells. Phospholipase C, TPA, and vanadate did not protect L cells against the conjugate. It is concluded that toxin in the conjugate enters L cells by a route which involves low pH, but which is not identical to that in Vero cells.     

Damage photosensitized by chlorine e6 in bacteria with various defects of the DNA repair system

The goal of the work was to study the sensitivity of isogenic Escherichia coli cells differing in their ability to mediate DNA repair steps to the action of visible light sensitized by chlorine e6. Cells incapable of excision repair as well as those deficient in post-replicative recombination DNA repair were found to be much more sensitive to the combined action of visible light and chlorine e6 as compared to cells whose genes responsible for DNA repair were not damaged. The results indicate that visible light damages bacterial DNA in the presence of chlorine e6.     

Selectivity of accumulation of chlorine e6 derivatives in blood leukocytes

The uptake of chlorine e6 derivatives by peripheral blood leukocytes was studied using a fluorescence-activated sorter. The analysis showed that the order of pigment uptake by leukocyte populations is the following: granulocytes > or = monocytes > lymphocytes. It was found that the accumulation of the pigments in the cell significantly varied. The level of chlorine e6 dimethyl ester accumulated by cells was found to be 15 times higher than that of chlorine e6. It was assumed that the differences in pigment uptake by different types of blood cells are due to structural and morphological features of leukocytes. The data obtained may be useful in developing the new methods of photodynamic therapy.     

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method for the fine-structural demonstration of lectin receptors

A new two-step method using an Fc-fragment/ferritin conjugate as a marker for the visualization of lectin-binding sites on neuronal and other cell membranes is described. In this study of rat synaptosomes, three lectins were tested: concanavalin A, mistletoe lectin I and wheat germ agglutinin. The specificity of the method was proved by control experiments.     

Lectin affinity electrophoresis for the separation of fluorescently labeled sugar derivatives.

Lectin affinity electrophoresis was applied to the separation of charged, fluorescent conjugates of disaccharides. Four fluorescent conjugates were prepared by reductive amination of alpha-D-Man-(1----3)-D-Man, alpha-D-Gal-(1----4)-D-Glc, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc in the presence of 7-amino-1,3-naphthalenedisulfonic acid. These charged fluorescent-disaccharide conjugates all have identical molecular weight and in the absence of conconavalin A lectin failed to separate either by agarose or by polyacrylamide gel electrophoresis. In the presence of either free or immobilized concanavalin A, agarose gel electrophoresis and polyacrylamide gel electrophoresis could separate the fluorescent conjugate of alpha-D-Man-(1----3)-D-Man from that of alpha-D-Gal-(1----4)-D-Gal, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc.     

A method for the fine-structural demonstration of lectin receptors

Summary A new two-step method using an Fc-fragment/férritin conjugate as a marker for the visualization of lectin-binding sites on neuronal and other cell membranes is described. In this study of rat synaptosomes, three lectins were tested: concanavalin A, mistletoe lectin I and wheat germ agglutinin. The specificity of the method was proved by control experiments.     

The distribution of chlorine e6 and its derivatives in HeLa cells studied using luminescence microscopy

The influence of the chemical structure of porphyrin pigments on their accumulation and localization in HeLa cells has been examined by the scanning fluorescence microphotometry. It has been found that the replacement of carboxyl groups of chlorine e6 for methyl and amino groups has no influence on the pigment distributions in cells. All the pigments are bound by cell membrane structures. The chemical modification of chlorine e6 structure is essential for the ability of pigment to be accumulated by cells that can be used to increase the efficiency of cancer phototherapy. The charge and hydrophobic properties of pigment molecules are of great importance for accumulating porphyrin sensitizers by cells.     

Formation and activity of covalent conjugates of poliovirus and ligands binding to cell surface structures

Disulfide-linked conjugates of poliovirus with streptavidin or concanavalin A were formed and the binding of the conjugates to mouse L cells that lack natural poliovirus receptors was studied. The conjugate with streptavidin was specifically bound to biotinylated L cells, but not to unmodified L cells. The conjugate with conA was bound to L cells in the absence of, but not in the presence of alpha-methyl mannoside. Incubation of L cells with bound conjugates did not produce virus, although the conjugates were highly infectious in HeLa cells, containing natural poliovirus receptors. This suggests that the artificially bound virus was unable to penetrate the L cells and start replication. The possibility that binding of the virus to the natural receptor is required for efficient infection is discussed.     

Conversion of pyridylamino sugar chains to 1-amino-1-deoxy derivatives, intermediates for tagging with fluorescein and biotin.

Pyridylamino (PA) derivatives of sugar chains were converted to 1-amino-1-deoxy derivatives. PA-lactose as a model compound was reduced with hydrogen, then treated with hydrazine. The product obtained was identified as 1-amino-1-deoxylactitol by mass spectrometry and chromatography with 1-amino-1-deoxylactitol as standard. PA-N-acetylglucosamine was converted to 1-amino-1-deoxy-N-acetylglucosaminitol under the same conditions. As an application, Man alpha 1-6(Man alpha 1-3)Man alpha 1- 6(Man alpha 1-2Man alpha 1-3)-Man beta 1-4GlcNAc beta 1-4GlcNAc-PA was converted to the 1-amino-1-deoxy derivative, which was further derivatized with fluorescein isothiocyanate or biotin sulfo-N-hydroxy-succinimide ester. Binding of these derivatives to concanavalin A dot-blotted on a nitrocellulose membrane was confirmed by fluorescence and by streptavidin-peroxidase conjugate. This conversion allowed replacement of the PA-group in PA-sugar chains which can be easily purified from glycoconjugates.     

Steroid nitrogen compounds. 3. A general method for introduction of O, N, S and other functionality into position-5 of cholestanes. Conjugate addition of nucleophiles to 6-nitrosocholesteryl acetate generated in situ from the 5 -chloro-6-oxime

The conjugate addition of organic and inorganic nucleophiles to 6-nitrosocholesteryl acetate (II) generated in situ from 3β-acetoxy-5-chloro-6-hydroxyimino-5α-cholestane (I) has been shown to give high yields of 5α-substituted 6-oximes (IIIa-h) under mild reaction conditions. Nucleophiles include ethanethiol, ethanol, methylamine, ammonia, nitrite, cyanide, thiocyanate and azide. Validity of the proposal that the replacement of chlorine atom of α-chlorooximes by nucleophiles proceeds via an elimination-addition mechanism through nitrosoolefins has been confirmed by the isolation and conversion of II into methoxy oxime (III, X = OMe) by the treatment with methanol. Due to the steric hindrance exerted by α-hydrogen atoms at λ-positions, bulky nucleophiles, which have been reported to react with α-chlorooximes, failed to react with the chlorooxime (I). Effects of 5α-substituents upon the signal position of the 3α-proton are described.     

Surface plasmon resonance based pesticide assay on a renewable biosensing surface using the reversible concanavalin A monosaccharide interaction

A competitive immunoassay based on surface plasmon resonance (SPR) for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The novelty of the assay is based on the regeneration of the chip surface by the reversible interaction between monosaccharide (D-glucose) and lectin (Concanavalin A). Concanavalin A-2,4-D conjugate was chemically synthesized, purified and used for binding to the SPR chip modified with covalently bound alpha-D-glucose. The interaction between anti-2,4-D antibody and the surface-bound concanavalin A-2,4-D conjugate was monitored by surface plasmon resonance and the response was used for the quantification of 2,4-D. The dynamic range of the calibration curve was between 3 and 100 ng/ml. The demonstrated principle of surface regeneration based on the reversible sugar-lectin interaction may be of more general applicability in immunoassays.     

Endogenous biotin‐binding proteins: an overlooked factor causing false positives in streptavidin‐based protein detection

Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin‐based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin‐associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin‐based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.     

Essential role of neutrophil mobilization in concanavalin A-induced hepatitis is based on classic IL-6 signaling but not on IL-6 trans-signaling

Neutrophil depleted mice are protected from concanavalin A-mediated hepatitis, showing that neutrophils are critical for cellular liver damage. Interleukin-6 has pro- and anti-inflammatory properties and mediates neutrophil recruitment in diseases such as rheumatoid arthritis. In classic signaling, interleukin-6 binds to the membrane-bound interleukin-6-receptor and initiates signaling via gp130. In interleukin-6 trans-signaling, the agonistic soluble interleukin-6-receptor can form a soluble interleukin-6/interleukin-6-receptor complex and stimulate cells which only express gp130 but no interleukin-6-receptor. Interleukin-6 trans-signaling was shown to be important for liver regeneration and development of liver adenomas. Here, we show that blocking classic interleukin-6 signaling but not interleukin-6 trans-signaling reduced concanavalin A-induced liver damage in mice, with reduced liver STAT3 phosphorylation and liver neutrophil accumulation. However, the level of neutrophil-attracting chemokine KC is only reduced by inhibition of interleukin-6 trans-signaling. Analysis of circulating neutrophils after concanavalin A challenge revealed that classic interleukin-6 signaling is required for the mobilization of blood neutrophils. Reduced neutrophil infiltration was accompanied by increased levels of hepatoprotective monocyte chemoattractant protein-1 and reduced level of hepatodestructive interleukin-4. Abrogated classic interleukin-6 signaling in concanavalin A-mediated hepatitis exhibited liver-protective effects indicating that interleukin-6 classic but not interleukin-6 trans-signaling is responsible for liver damage. Classic interleukin-6 signaling is required to mount an efficient neutrophilia during concanavalin A-induced immune response, which might have clinical implications in the regard that blocking global interleukin-6 signaling pathways is a treatment option in different chronic inflammatory diseases.     

Detection of human hematopoietic differentiationrelated glycoproteins: The Western enzyme-linked lectin analysis

A technique is introduced (Western enzyme-linked lectin analysis, WELLA) for detecting lectin-reactive cellular glycoproteins after separation on the basis of molecular weight in sodium dodecyl sulfate (SDS) polyacrylamide gels. Lectin-reactive glycoproteins are detected on Western transfers by reaction with lectin-peroxidase conjugates followed by development with hydrogen, peroxide and 4-chloro-1-naphthol which forms a purple-gray precipitate. WELLA is more rapid, more sensitive, and the bands are highly reproducible and better resolved than those obtained, by autoradiography of fluorography.Using this technique, we have detected human differentiation-related glycoproteins on cells of different hematological lineages. Both wheat germ agglutinin-peroxidase (WGA-P) and concanavalin A-peroxidase (ConA-P) detected distinct glycoprotein patterns on isolated peripheral blood platelets, lymphocytes, monocytes, erythrocytes and granulocytes. WGA-P detected numerous similarities between immature myeloid cells isolated from bone marrow and acute myelogenous leukemia cells, including major glycoproteins at 20 and 25 kDa. ConA-P detected a similar pattern of glycoproteins between isolated peripheral blood lymphocytes and T-cell acute lymphoblastic leukemia (T-ALL) cells. The T-ALL cells, however, had a major 200 kDa glycoprotein not present on lymphocytes. WGA-P also showed nearly identical patterns between the lymphocytes and the T-ALL cells, but detected prominent 200 and 250 kDa glycoproteins on the T-ALL cells which were absent from the lymphocytes. We have also detected polymorphic differences in the glycoproteins on lymphocytes from normal donors in the range of 95-100 kDa using ConA-P.Abbreviations WELLA Western enzyme-linked lectin analysis - SDS sodium dodecyl sulfate - BSA bovine serum albumin - PVP polyvinylpyrrolidone - PBS phosphate-buffered saline - AML acute myelogenous leukemia - ALL acute lymphocytic leukemia - WGA wheat germ agglutinin - Con A concanavalin A - WGA-P wheat germ agglutinin-peroxidase conjugate - ConA-P concanavalin A-peroxidase conjugate     

Localization of carbohydrate components in rat colon with fluoresceinated lectins

Cryostat sections of rat descending colon were studied by fluorescence microscopy after exposure to conjugates of fluorescein isothicoyanate with lectins from Glycine max (soybean), Triticum vulgaris (wheat germ), Ricinus communis (castor bean), Ulex europaeus, (gorse), Dolichos biflorus (horse gram) and Canavalia ensiformis (concanavalin A) (Jack bean). No two lectins showed identical patterns of fluorescence. FITC-conjugates of soybean and D. biflorus lectins reacted strongly with the mucus present in the crypt lumens and with the surface (as well as cytoplasm) of the epithelial cells suggesting that these sites are rich in terminal, non-reducing, N-acetylgalactosamine residues. Wheat germ, R. communis, U. europaeus and concanavalin A-FITC conjugates did not stain mucus but showed fluorescence in the cytoplasm of absorptive cells as well as in the lamina propria and submucosa. The FITC-R. communis conjugate also reacted with structures in the apical portion of epithelial cells that may correspond to the Golgi apparatus.     

The role of tetrapyrrol photosensitizers in photoinduced variation of free radical characteristics of rat blood in endotoxic shock

The main goal of the present study was to evaluate the comparative effectiveness of tetrapyrrol photosensitizers (protoporphyrine IX and chlorine e6) in red (632.8 nm) and green (532.5) spectrum bands on rat blood free radical status, using the experimental model of endotoxic shock. Endotoxic shock was produced by intraperitoneal injection of lipopolysaccharide B. Irradiation effectiveness was estimated by leukocyte activation (measured with luminol-dependent chemiluminescence), superoxide dismutase activity of blood plasma (nitro blue tetrasolium assay) and lipid peroxidation (assay with cis-parinaric acid). It was found that laser irradiation has multidirectional effects on leukocyte activation, membrane lipid peroxidation and plasma SOD activity and all these effects were more pronounced in the case of endotoxic shock. Protoporphyrin was more effective in leukocyte activation and chlorine e6 demonstrated maximal effects on blood SOD activity.