Concanavalin A-induced Agglutination Research Articles

Effect of Treatment with Phospholipase A2, Colchicine, and β-Galactosidase on Agglutination of Rat Hepatocytes

The effects of phospholipase A2, colchicine, and β-galactosidase on concanavalin A-induced agglutination of viable hepatocytes isolated from normal and diabetic rats are reported. Phospholipase A2 (0.92 μg/mL), colchicine (400 μg/mL), and β-galactosidase (300 μg/mL) treatments caused a significant increase in the agglutination rate of hepatocytes. These findings suggest that phospholipase A2 treatment resulted in the unshielding of lectin receptors. Colchicine treatment seemed to release those receptors from cellular restraints which tend to separate and/or direct them. The promoting effect of β-galactosidase could be attributed to a decrease in repulsive forces due to a reduction in net negative charge density after removal of N-acetylneuraminic acid residues. Normal rat hepatocytes seem to be richer in galactosides, phospholipids, and the microfilament-microtubule network than their diabetic counterparts.

Studies on cell adhesion and concanavalin A-induced agglutination of Candida albicans after mannan extraction.

Candida albicans cells were treated with alkali and acid to extract preferentially the cell wall alpha-mannan. Cells were recovered at three stages, as extraction proceeded from mild to more extensive: Alk-1, Alk-2 and Alk + Acid. Yeast adhesion to human epithelial cells was then examined with an in-vitro adherence assay. Yeasts from all three stages of extraction adhered in significantly lower numbers to buccal mucosal cells than did unextracted yeasts. Adhesion was as low for Alk-1 cells as for those submitted to more complete mannan extraction. When yeast cells from all three stages were treated with Concanavalin A (Con A), a lectin probe with strong affinity for yeast alpha-mannans, and then subjected to the adherence assay, there was no significant change in adhesion. When yeast agglutinability by Con A was examined in tests with treated and untreated yeast cells, abundant agglutination occurred only with the untreated cells. However, Alk-1 cells, though lacking in adhesive capacity towards mucosal cells, showed significant agglutination. The results suggest that candidal adhesion is mediated by an alkali-soluble, mannan-containing moiety(ies) which appears to be lost early in the extraction process. Blockage of this moiety by Con A inhibits the adhesion of unextracted cells. Extracted cells lack this moiety but still possess enough structural mannan for Con A recognition and agglutination.

T-cell-fibroblast hybridoma deformability and concanavalin A-induced agglutination.

Cell adhesion influences many important immunological functions such as phagocytosis or T-cell-mediated cytotoxicity. Previous work suggested that the ease of inducing intercellular bonds (i.e. binding efficiency) and the difficulty to separate bound cells with mechanical forces (i.e. binding strength) might be parameters of different significances. The present report describes a study made on two T-lymphocyte/polyoma virus transformed fibroblast hybrid subclones (3D1c and 3D1n) with markedly different adhesive properties: indeed, 3D1c cells were at the same time more readily agglutinated with concanavalin A and more easily disagglutinated than 3D1n. In order to understand these differences, a systematic comparison of various properties of 3D1c and 3D1n cells was undertaken. The following parameters were studied: surface density of concanavalin A binding sites, surface electrostatic charge, hydrophobicity, fluorescence polarization measured on individual cells, and ability to spread on a flat substrate in response to volume or surface forces. It is concluded that cell deformability and/or spreading ability might be an important determinant of binding strength, but the factors governing binding efficiency remained incompletely understood. It is suggested that the methods described in the present report might help understanding differences between various tumor cell lines with different malignant potential.

Interaction of concanavalin A with spin-labeled glycolipid incorporated into liposomes.

Using a synthetic glycolipid derived from maltotetraose and a spin-labeled fatty acid, the lateral distribution and molecular motion of the spin-labeled glycolipid on phosphatidylcholine liposomes in the presence and absence of concanavalin A were examined. When the spin-labeled glycolipid was added to preformed egg yolk phosphatidylcholine-dicetyl phosphate (molar ratio, 10 : 1) liposomes, most of the spin-labeled glycolipid molecules could be incorporated into liposomes as shown by their concanavalin A-induced agglutination. Concanavalin A also caused a change in line width of the ESR signal of liposome-bound spin-labeled glycolipid, whereas the overall splitting value 2A parallel did not change significantly. It is suggested that the binding of glycolipid molecules to concanavalin A increased the interactions among the radicals of the probe but that the mobility of the acyl chain off glycolipids was not affected. These signal changes were also observed with succinyl-concanavalin A. However, in contrast to concanavalin A, no appreciable agglutination of liposomes could be induced by the latter concanavalin A derivative. Both the agglutination of liposomes and the change in line width of the ESR signal were completely inhibited by alpha-methyl-D-mannoside.

The effect of diabetes on hepatocyte plasma membrane fluidity and concanavalin A-induced agglutination

The techniques of fluorescence polarization and lectin-induced agglutination have been utilized to investigate the effects of the diabetic state on some of the dynamic properties of cell membranes. Hepatocyte plasma membranes from streptozotocin-induced diabetic rats exhibited a significant decrease in cholesterol and sialic acid with no alteration in phospholipid content. This membrane system also exhibited a decrease in fluorescence polarization, using the fluorescent probe, 1,6-di-phenyl-1, 3,5-hexatriene, suggesting an increase in membrane fluidity over the value observed in normal hepatocytes. When normal hepatocytes were incubated in the presence of the lectin, concanavalin A (ConA), no significant agglutination was observed. In contrast, hepatocytes from diabetic rats which exhibited a slightly decreased lectin-binding capacity underwent extensive agglutination. In addition, normal hepatocytes which were pretreated with 0.1 mM tetracaine also underwent extensive agglutination with no measurable increase in lectin-binding capacity. These results suggest that altered membrane lipid fluidity and/or cytoskeletal organization may have a profound effect on cell surface dynamics and could result in the uncoupling of the insulin receptor complex from the membrane-associated effector system(s), a defect which may play a role in the problem of insulin resistance observed in some forms of diabetes.

Role of cell surface receptor mobility in concanavalin A-induced agglutination of Novikoff hepatoma and normal rat liver cells.

The relationshio between Con A-receptor mobility and Con A-induced agglutination of Novikoff hepatoma and normal rat liver cells was investigated. Novikoff cells, incubated with fluorescein-labelled Con A at 3 degrees C displayed uniform, ring-like surface fluorescence. Increasing the temperature of the cells to 37 degrees C caused capping of Con A receptors in approximately 65% of the cells, a phenomenon that could be prevented by prefixing the cells with glutaraldehyde. In spite of these variations in Con A-receptor distribution, Con A-induced agglutination was remarkably constant over a temperature range from 3 to 37 degrees C. In contrast to Novikoff cells, normal hepatocytes displayed a uniform, ringlike surface fluorescence at both 3 and 37 degrees C. No capping was observed. However, hepatocytes, similar to Novikoff cells, were agglutinable by low concentrations of Con A. These findings indicate that, in this model system, Con A-induced cytoagglutination is not dependent upon long-range lateral mobility of Con A receptors. The qualitative differences in the lateral mobility of cell-surface Con A receptors of normal and malignant rat liver cells may represent a marker for neoplastioc transformation during hepatocarcinogenesis, adaptation to growth in ascitic form, or progression of a tumour to a more malignant state.

Agglutination kinetics of normal and diabetic adult rat hepatocytes.

This paper describes Concanavalin A-induced agglutination of viable rat hepatocytes obtained by collagenase perfusion from normal and streptozocin-treated diabetic rats. An irreversible cell-to-cell agglutination model is proposed to explain hepatocyte flocculation. The rate of agglutination is concentration dependent with respect to Concanavalin A, and is twice as fast in normal as compared to diabetic hepatocytes. Sticking probability constants ranging from 18.48 x 10(7) to 4.6 x 10(7) cm-1 . hepatocyte-1 and 8.32 x 10(7) to 2.31 x 10(7) cm-1 . hepatocyte-1 are calculated as a measure of agglutination for normal and diabetic cells respectively. These findings suggest that the cytoplasmic membranes of normal cells possess agglutinin receptors which are more numerous and/or differently arranged than those existing in diabetic cells.

Inhibition of concanavalin A-induced agglutination of Novikoff tumor cells by cytochalasins and metabolic inhibitors: Role of cell-surface morphology and the distribution of concanavalin A receptors

Previous drug studies have suggested that concanavalin A (ConA)-induced cytoagglutination may be influenced by a system of contractile microfilaments. The present study was undertaken to determine the effects of microfilament-active drugs (cytochalasins B and D) (CB and CD) and inhibitors of ATP formation (NaN 3 and 2,4-dinitrophenol (DNP)) on ConA-induced agglutination of Novikoff cells and to investigate the mechanism(s) whereby these agents alter the surface properties of cells. The study described herein demonstrated that 1. 1. CB or CD inhibit cytoagglutination at concentrations above 1 or 0.1 μg/ml, respectively. 2. 2. NaN 3 and DNP both inhibit cytoagglutination, but DNP is more effective and specific. 3. 3. Combinations of metabolic inhibitors (NaN 3 or DNP) with CB lead to a greater reduction of cytoagglutination than with either agent alone. 4. 4. Maximal (>95%) cytoagglutination is still achieved in the presence of CB, CD, NaN 3, DNP, or combinations of these drugs. 5. 5. None of the drugs tested induced or allowed the redistribution of ConA receptors as measured by ferritin-ConA labeling. 6. 6. Both CB and CD induced the appearance of numerous blebs (zeioses) at the cell periphery, whereas metabolic inhibitors did not. 7. 7. The concentration of either CB or CD required to alter cell-surface morphology paralleled the concentration necessary to inhibit cytoagglutination. These results suggest that the cytochalasins inhibit ConA-induced agglutination of Novikoff cells by their effect on cell-surface morphology, rather than by their effects on the topographical distribution of cell-surface lectin receptors, and that metabolic inhibitors reduce agglutinability by a different mechanism than the cytochalasins.

Biochemical Characteristics of Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) has a variable clinical course and there is a great need of new prognostic laboratory parameters in this disease. 24 CLL patients were subjected to routine haematological and clinical investigation. The leukaemic cells were analyzed for surface immunoglobulins, complement and sheep red blood cell receptors, Concanavalin A-induced agglutination, cytoplasmic glucocorticoid receptor and for proliferative activity by measurement of tritiated thymidine incorporation. The surface markers studied indicated that all cases were of B-cell origin. Only 5 of 23 patients studied had normal serum immunoglobulin levels. These cases showed a nonprogressive disease. 8 patients had increased infection tendency, all of whom had subnormal IgG levels; 4 of them also had subnormal IgA and IgM and 2 had subnormal IgA levels. 5 out of 6 patients with progressive disease and 3 of 11 with nonprogressive disease had an increased proliferative index, indicating a correlation between this parameter and disease progression. ConA agglutinability was not correlated to disease activity. Cells from 17 of 22 patients showed measurable amounts of glucocorticoid receptor. The 5 patients lacking this receptor had inactive disease.

Spontaneous and concanavalin A-induced agglutination of chick-embryo neuronal cells

Concanavalin A-induced cell agglutination of different neuronal cells from the spinal cord and cerebral cortex of the chick reaches maximum values between the 9th and 11th day of embryogenesis. With further development, i.e. with increasing age, cell agglutination decreases dramatically. However, Concanavalin A binding sites were still found during the period of decreased agglutination. A pronounced difference between spontaneous and Concanavalin A-induced agglutination can be detected. The latter leads to the formation of special contact regions, where the cells develop pseudopods and remain rather motile. The results support the idea that Concanavalin A-induced agglutination, in contrast to spontaneous agglutination, is an active process of cellular interaction which is dependent on receptor mobility and related to surface movement and cell motility. Therefore this type of agglutination may be restricted to early motile stages of developing neuronal cells.

Concanavalin A-induced agglutination of Naegleria.

Concanavalin A (Con A) agglutinated all Naegleria gruberi strains tested but did not agglutinate any N. fowleri strains tested. Agglutination was time and temperature dependent and Con A concentration and ameba concentration dependent over certain ranges. Agglutination increased to maximum up to 1 h incubation with Con A. At least 1 X 10(6) amebae/ml were needed for maximum agglutination, and Con A concentrations higher than 100 microgram/ml did not appreciably increase agglutination. Incubation of 4 degrees C or with 10 mM alpha-methyl-D-mannoside inhibited agglutination of N. gruberi. These data indicate a difference in polysaccharide structure of cell membranes of N. fowleri and N. gruberi.

Surface properties related to concanavalin A-induced agglutination. A comparative study of several Entamoeba strains.

Pathogenic strains of Entamoeba histolytica are more easily agglutinated with concanavalin A (Con A) than strains isolated from human asymptomatic carriers. All three pathogenic strains studied here were found to agglutinate with low concentrations of Con A in contrast to various nonpathogenic axenic strains of amebas, characterized by their ability to grow at room temperature. Our present observations suggest that the extreme susceptibility of pathogenic strains of E. histolytica to agglutinate with Con A is related to their higher capacity for lectin binding and to their lack of detectable repulsive charges at the cell surface. The amount of fluorescein-tagged Con A bound to the surface was much higher in pathogenic strains. Only nonpathogenic strains showed a detectable negative surface charge as studied both by means of cell microelectrophoresis and by labeling cells with cationized ferritin at 0 degrees C. The mobility of surface Con A receptors estimated as the percentage of caps was comparable in all strains. Results of one strain cultured in axenic and monoxenic conditions suggested that bacteria can modify the behaviour of E. histolytica trophozoites by altering surface properties of the amebas.

A lipopeptidophosphoglycan from Trypanosoma cruzi (epimastigota). Isolation, purification and carbohydrate composition.

A lipopeptidophosphoglycan was extracted from epimastigote forms of Trypanosoma cruzi by phenol (44%) treatment of sonicated cells. The substance was purified from other glycoproteins and nucleic acid as follows: ethanol fractionation. Bio-Gell P-150 column chromatography in the presence of 0.1% sodium dodecyl sulfate, extraction with chloroform/methanol/water (10 : 10 : 3) and precipitation of the pure compound by methanol. The substance migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis stained with periodic acid-Schiff and Coomassie blue. In the absence of sodium dodecyl sulfate very little or no migration was observef aggregates. In such gels a Sudan Black positive reaction coincident with the periodic acid-Schiff positive banc was obtained. Neutral sugars (60%, by phenol-sulfuric acid assay) were analysed by paper chromatography and gal-liquid chromatography. The following ratio was found: mannose : galactose : glucose = 35 : 22 : 1. Glucosamine, identified by paper chromatography, was colorimetrically estimated (0.8%). Sialic acid was not detected. Analysis by the biuret method gave 9.5% protein. All phosphorus present (2%) was released by hydrolysis, thus apparently excluding the possibility of an alkyl phosphonic acid as a structural component. Fatty acids were detected by thin layer chromatography in a hexane extract of the acid hydrolysate. Gas-liquid chromatography of the esterified mixture showed that the main component had the same retention time as palmitic acid methyl ester. The infrared spectrum was consistent with the general structure and indicated the presence of alpha-glycopyranosyl linkages. Low concentrations of the lipopeptidophosphoglycan were able to inhibit the concanavalin A-induced agglutination of epimastigotes.

Concanavalin A-Induced Agglutination of Cells Infected with Inactive Herpes Simplex Virus

Concanavalin A-Induced Agglutination of Cells Infected with Inactive Herpes Simplex Virus

Concanavalin A-induced agglutination of Acanthamoeba.

VIRULENT strains of Acanthamoeba produce cytopathic effects in mammalian tissue cultures1–3, and cause acute meningoencephalitis in laboratory animals after intranasal inoculation1. In humans, Acanthamoeba seem to produce cerebral lesions in defence-weakened individuals4–7. We have begun studies of the cell surface of Acanthamoeba since virulent strains must make direct contact with a host cell before initiation of degenerative changes in mammalian tissue cultures2,3. The sensitivity of avirulent and virulent strains to concanavalin A (con A)-induced agglutination was investigated first as there was no evidence that carbohydrate-containing components were exposed at the cell surface of Acanthamoeba. Our results indicate that both avirulent and virulent Acanthamoeba were sensitive to con A-induced agglutination. In contrast to the results of studies of avirulent and virulent Entamoeba histolytica8, however, the avirulent strain was more sensitive to con A.