Induction and regulation of neuraminidase synthesis in Arthrobacter sialophilus.

A variety of N-acetylneuraminic acid (AcNeu) derivatives and analogs were examined as inducers of the extracellular neuraminidase of Arthrobacter sialophilus. Neuraminidase inductions were primarily studied with tryptone-yeast extract-grown cells after washing and resuspension in a defined replacement medium. The addition of readily metabolizable carbon sources to the latter, such as 0.1% casein hydrolysate, glutamate, or glucose, enhanced enzyme synthesis. Enzyme appearance occurred after a lag in the uptake of inducers, suggesting the participation of a co-inducible transport system. Neuraminidase formation during exponential growth in the presence of AcNeu ceased after depletion of this end product from the medium. It was found, besides AcNeu, that its methyl ester, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid methyl ester are each active inducers, whereas beta-anomers of AcNeu-ketosides are not. These results, in comparison to known enzyme specificity, have revealed significant differences and parallels between the inductive and catalytic processes for neuraminidase. In particular, it would appear that the free carboxylate and oxygenation at C-2 of AcNeu, essential for enzyme catalysis with traditional AcNeu substrates, are not necessary for induction and, furthermore, that transition state analogs can specifically induce this enzyme. The failure to observe catabolite repression in this system is discussed in relation to the intermediary metabolism of the genus Arthrobacter.     

Subcellular localization and tissue distribution of sialic acid-forming enzymes. N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase.

The activities of N-acetylneuraminate 9-phosphate synthase and N-acetylneuraminate 9-phosphatase, the two enzymes involved in the final steps of the biosynthetic pathway of N-acetylneuraminic acid, were measured with the substrates N-acetyl[14C]mannosamine 6-phosphate and N-acetyl[14C]neuraminic acid 9-phosphate respectively. Subcellular localization studies in rat liver indicated that both enzymes are localized in the cytosolic fraction after homogenization in sucrose medium. To test the possibility of misinterpretation due to the hydrolysis of N-acetylneuraminic acid 9-phosphate by non-specific phosphatases, the hydrolysis of various phosphate esters by the cytosolic fraction was tested. Only p-nitrophenyl phosphate was hydrolysed; however, competition studies with N-acetylneuraminic acid 9-phosphate and p-nitrophenyl phosphate indicated that two different enzymes were involved and that no competition existed between the two substrates. In various other rat tissues N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase activities were detected, suggesting that N-acetylmannosamine 6-phosphate is a general precursor for N-acetylneuraminic acid biosynthesis in all the tissues studied.     

The biosynthesis of N-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of N-acetylneuraminic acid

The biosynthesis of N-glycoloylneuraminic acid in fractionated porcine submandibular glands was investigated. The following substrates: [3H]N-acetylmannosamine, free [14C]N-acetylneuraminic acid, CMP-[14C]N-acetylneuraminic acid, [14C]N-acetylneuraminic acid linked alpha(2----3) to galactose residues, or alpha(2----6) to Gal-beta(1----4)-GlcNAc residues of porcine submandibular mucin and [14C]N-acetylneuraminic acid linked alpha(2----6) to GalNAc residues of ovine submandibular gland mucin were incubated, in the presence of cofactors, with the soluble protein, heavy membrane and microsomal fractions of porcine submandibular glands. Radio thin-layer chromatographic analysis revealed that only one substrate, CMP-[14C]N-acetylneuraminic acid, was hydroxylated. The product was identified as CMP-[14C]N-glycoloylneuraminic acid by (i) co-chromatography with non-radioactive CMP-N-glycoloylneuraminic acid standard, (ii) acid hydrolysis to free [14C]N-glycoloylneuraminic acid, (iii) alkaline hydrolysis to yield N-glycoloylneuraminic acid and 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid and (iv) transfer of [14C]N-glycoloylneuraminic acid to asialo-fetuin by sialyltransferase. 85% of CMP-N-acetylneuraminic acid hydroxylase activity was present in the soluble protein fraction, with small amounts of activity in the two particulate fractions. The CMP-N-acetylneuraminic acid hydroxylase in the soluble protein fraction had an absolute requirement for Fe2+ ions and a reducing cofactor. NADPH and NADH were by far the most effective cofactors, smaller amounts of hydroxylation could, however, be supported by ascorbic acid and 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin.     

A monoclonal antibody to free N-acetylneuraminic acid

A monoclonal antibody (70-A) to free N-acetylneuraminic acid was obtained by immunizing mice with its synthetic beta-glycoside, sodium O-[(5-acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-2- nonulopyranosyl)onate]-(2----3)-1,2-di-O-tetradecyl-sn-glyce rol, followed by fusing the isolated spleen cells with mouse myeloma cells and cloning positive fusions. 70-A reacted with various synthetic beta-glycosides of N-acetylneuraminic acid and also with cytidine-5'-monophosphate-N- acetylneuraminic acid, known as its sole naturally occurring beta-glycoside. The inhibition assay showed that N-glycolylneuraminic acid had slightly lower reactivity than N-acetylneuraminic acid, but other monosaccharides tested, such as N-acetylglucosamine, N-acetylgalactosamine or N-acetylmannosamine, had no reactivity toward 70-A. Reactivity of 70-A with free N-acetylneuraminic acid was confirmed by measuring the specific binding of N-[14C]acetylneuraminic acid to the antibody. The association constant of 70-A with N-acetylneuraminic acid was determined to be 5.96.10(4) M-1 by equilibrium dialysis.     

Distribution of free N-acetylneuraminic acid in rat organs

The concentration of free N-acetylneuraminic acid in various rat organs was estimated by gas chromatography/mass spectrometry. Its concentration was in the range of 3.95 to 104.72 micrograms/g wet tissues, being particularly high in the endocrine glands. The ratio of free N-acetylneuraminic acid to total N-acetylneuraminic acid varied from 0.031 to 0.183, being especially high in the adrenal gland (0.181) and heart (0.183).     

Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase

Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme.     

A factor in animal tissues that causes sialyl residues of mucoproteins to react as free sialic acid in the Warren assay

1. The mucin of the Cowper's gland of the boar is a sialomucoprotein similar to submaxillary-gland mucin. When a solution of either mucin has been incubated for 5min or less with a particulate fraction from homogenized uterine endometriumplus-myometrium of the rabbit, 10-20% of sialyl residues (N-acetylneuraminic acid) give a positive Warren reaction for free N-acetylneuraminic acid. The particulate fraction is devoid of neuraminidase and no free (diffusible) N-acetylneuraminic acid appears during incubation. The factor that catalyses the formation of directreading non-diffusible N-acetylneuraminic acid occurs also in liver, kidney and intestinal mucosa of the rabbit. The factor is present in very small (;microsomal') particles and has not yet been solubilized. Homogenates of boar Cowper's gland contain both factor and mucin; thus direct-reading non-diffusible N-acetylneuraminic acid appears when such homogenates are stored. 2. Under optimum conditions 1mg of uterine protein catalyses the formation of 0.05-0.1mumol of direct-reading non-diffusible N-acetylneuraminic acid/min. This activity is considerably higher than the neuraminidase activities of comparable homogenates of animal tissues or of liver lysosomes. The factor is thermostable and its activity shows little variation within (i) the pH range 3-10, (ii) the temperature range 20-37 degrees C. Activity is inhibited strongly by 2,2'-bipyridyl and by ammonium pyrrolidine dithiocarbamate but is unaffected by EDTA. Its action can be simulated by low concentrations of Fe(2+). From this it may be inferred that the factor is a protein-bound from of bivalent iron. A number of pure iron-containing proteins and haemoproteins were completely inactive. The following substrates were not sources of direct-reading non-diffusible N-acetylneuraminic acid: methoxyneuraminic acid, sialyl-lactose, brain gangliosides, and sialoproteins in which N-acetylneuraminic acid is linked to galactose residues. 3. It is proposed that the factor (or Fe(2+)) reacts with the mucin in a manner that renders the C-4-C-5 bond of sialyl residues susceptible to periodate oxidation.     

Preparation and properties of sialomucopolysaccharides obtained from rat brain

Sialomucopolysaccharides were released from the defatted protein residue by the proteolytic action of papain after extraction of rat whole brains with chloroform-methanol (2:1, v/v). Further purification is achieved by dialysis to remove low-molecular-weight fragments and by precipitation of nucleic acids and glucuronic acid-containing mucopolysaccharides by treatment with cetylpyridinium chloride. Gel filtration of the sialomucopolysaccharides through Sephadex G-200 removes the major portion of the impurities that absorb light in the ultraviolet region. The sialomucopolysaccharides were fractionated on DEAE-Sephadex to yield a population of sialomucopolysaccharides that show an increase in N-acetylneuraminic acid content and a decrease in fucose content as the concentration of chloride required to elute the individual components is increased. On gel filtration on Sephadex G-200, those sialomucopolysaccharide molecules rich in N-acetylneuraminic acid and poor in fucose appear to be larger molecules than those rich in fucose and poor in N-acetylneuraminic acid. A structure is proposed in which all sialomucopolysaccharide molecules are assumed to possess the same repeating unit consisting of hexosamine and hexose. The molecules differ from each other in the number of fucose and N-acetylneuraminic acid residues attached to the basic structure. Most of the hexosamine is present as glucosamine, although one fraction was obtained that appeared to contain galactosamine. Most of the hexose present is accounted for as galactose and mannose, although small amounts of glucose were found in some fractions. Methods of analysis for the N-acetylneuraminic acid and hexosamine components of the sialomucopolysaccharides were defined.     

Synthesis of rat-liver lactate dehydrogenase and characterization of its mRNA

Salla disease is a lysosomal storage disorder of unknown etiology, characterized biochemically by increased urinary excretion of N-acetylneuraminic acid. This compound has now been shown to occur in abnormally large amounts in liver and cultured skin fibroblasts from these patients. Quantification of N-acetylneuraminic acid was performed using a new gas-chromatography/mass spectrometric single-ion method which is sensitive and specific. No abnormalities in the activity of several enzymes involved in sialic acid metabolism (N-acetylneuraminate:pyruvate lyase, neuraminidase, CMP-N-acetylneuraminate N-acylneuraminohydrolase and CTP:N-acyl-neuraminate cytidylyltransferase) were demonstrable. A possible explanation for the defect is a malfunctioning active transport of N-acetylneuraminic acid across the lysosomal membrane.     

N-acetylneuraminic acid and N-glycolylneuraminic acid in glycopeptides of colonic tumor and mucosa in rats treated with carrageenan and 1,2-dimethylhydrazine

N-linked glycopeptides were prepared from colonic tumor (adenocarcinoma) and mucosa in rats treated with carrageenan, an indigestible polysaccharide, and 1,2-dimethylhydrazine. Sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, obtained by acid hydrolysis of the glycopeptides were determined by HPLC. The N-acetylneuraminic acid/N-glycolylneuraminic acid ratio in colonic tumor was 25.2, while each treated mucosa had the values between 0.29 and 0.55. Thus, necessity which observes the qualitative change of sialic acid in malignant transformation was suggested.     

Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst

Bioprocess and Biosystems Engineering - N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) were immobilized for synthesis of N-acetylneuraminic acid (Neu5Ac) on three...     

The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain

Protein-bound N-acetylneuraminic acid and hexosamine, including the sialomucopolysaccharides, occur mainly in the least dense particles sedimented in the microsomal fraction from rat whole brain. Particles rich in protein-bound N-acetylneuraminic acid and hexosamine are also found in the subcellular fraction separated as a layer between 0.8m- and 1.2m-sucrose after centrifuging the crude mitochondrial preparation in a density gradient. This distribution is similar to that of the gangliosides and suggests an association of all of these substances in the same subcellular structures. It is postulated that the sialomucopolysaccharides, as well as the gangliosides, are components of cell membranes. Evidence is presented that indicates that there are quantitative differences between distribution of the gangliosides on the one hand, and protein-bound N-acetylneuraminic acid and hexosamine on the other. The ratio of protein-bound N-acetylneuraminic acid (and hexosamine) to gangliosidic N-acetylneuraminic acid (and hexosamine) present in individual subcellular fractions obtained by density-gradient centrifugation tends to increase with increasing particle density. Exposure of the crude mitochondrial fraction to osmotic ;shock' before density-gradient centrifugation causes a shift of the protein-bound N-acetylneuraminic acid and gangliosides to the less dense fractions. In some experiments, a selective shift of the protein-bound N-acetylneuraminic acid was observed.     

The influence of N-acetylneuraminic acid on the properties of human orosomucoid.

Little is known of the relationships that may exist among the three principal functionalities of glycoproteins. Orosomucoids of closely defined N-acetylneuraminic acid content were examined for evidence of influence of N-acetylneuraminic acid content on the physical properties of the glycoprotein. Fluorescence spectroscopy gave no indication of conformational change in the protein core upon desialylation. Small changes in the chromatographic partition coefficient, sigma, and thermal stability, Td, are interpreted to reflect loss of water of hydration and increased glycan stem-protein interaction without a major repositioning of the chains. Ligand-binding measurements indicate no alteration in the hydrophobic binding domain and a possible interaction between chlorpromazine and N-acetylneuraminic acid. All changes seen are progressive and occur through a region where changes in biological activity are not found. It is suggested that the dependence of biological activity on N-acetylneuraminic acid content in orosomucoid reflects, not coupled changes in protein conformation, but a charge-density-related interaction such that, below a contribution of four or five N-acetylneuraminic acid residues, activity is modified.     

Purification and properties of N-acetylneuraminate lyase from Escherichia coli

N-Acetylneuraminate lyase [N-acetylneuraminic acid aldolase EC 4.1.3.3] from Escherichia coli was purified by protamine sulfate treatment, fractionation with ammonium sulfate, column chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA 44, and preparative polyacrylamide gel electrophoresis. The purified enzyme preparation was homogeneous on analytical polyacrylamide gel electrophoresis, and was free from contaminating enzymes including NADH oxidase and NADH dehydrogenase. The enzyme catalyzed the cleavage of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate in a reversible reaction. Both cleavage and synthesis of N-acetylneuraminic acid had the same pH optimum around 7.7. The enzyme was stable between pH 6.0 to 9.0, and was thermostable up to 60 degrees C. The thermal stability increased up to 75 degrees C in the presence of pyruvate. No metal ion was required for the enzyme activity, but heavy metal ions such as Ag+ and Hg2+ were potent inhibitors. Oxidizing agents such as N-bromosuccinimide, iodine, and hydrogen peroxide, and SH-inhibitors such as p-chloromercuribenzoic acid and mercuric chloride were also potent inhibitors. The Km values for N-acetylneuraminic acid and N-glycolylneuraminic acid were 3.6 mM and 4.3 mM, respectively. Pyruvate inhibited the cleavage reaction competitively; Ki was calculated to be 1.0 mM. In the condensation reaction, N-acetylglucosamine, N-acetylgalactosamine, glucosamine, and galactosamine could not replace N-acetylmannosamine as substrate, and phosphoenolpyruvate, lactate, beta-hydroxypyruvate, and other pyruvate derivatives could not replace pyruvate as substrate. The molecular weight of the native enzyme was estimated to be 98,000 by gel filtration methods. After denaturation in sodium dodecyl sulfate or in 6 M guanidine-HCl, the molecular weight was reduced to 33,000, indicating the existence of 3 identical subunits. The enzyme could be used for the enzymatic determination of sialic acid; reaction conditions were devised for determining the bound form of sialic acid by coupling neuraminidase from Arthrobacter ureafaciens, lactate dehydrogenase, and NADH.     

Distribution of neuraminidase in Arthrobacter and its purification by affinity chromatography

Neuraminidase [sialidase, EC 3.2.1.18] was found to be widely distributed in bacteria belonging to Arthrobacter. Among these bacteria, Arthrobacter ureafaciens, A. oxydans, and A. aurescens produced relatively potent neuraminidase activities. For the production of this enzyme, not only colominic acid, a homopolymer of N-acetylneuraminic acid, but also N-acetylneuraminic acid, the reaction product of this enzyme, are effective as sources of carbon. An affinity adsorbent specific for neuraminidase was prepared by cross-linking colominic acid with soluble starch by means of epichlorohydrin. Neuraminidase from A. ureafaciens could be purified on this affinity column. The purified neuraminidase was shown to be free from protease, N-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. Aminoff's assay procedure for sialic acid was modified to avoid the centrifugation step. The modified procedure gave a higher molecular extinction coefficient.     

Gracilaria tikvahiae agglutinin. Partial purification and preliminary characterization of its carbohydrate specificity

A potent agglutinin of rabbit and sheep red blood cells, obtained from the red alga Gracilaria tikvahiae, was purified by ammonium sulfate fractionation, ion exchange, gel filtration, and hydroxylapatite chromatography. Human A and B blood group erythrocytes were also agglutinated, whereas human O blood group erythrocytes were not agglutinated. The hemagglutination titer was not significantly affected by the addition of EDTA or the divalent cations Ca2+, Mg2+, or Mn2+. The carbohydrate specificity was characterized by hemagglutination inhibition using various monosaccharides, glycoproteins, and glycopeptides. The results suggested that the agglutinin has affinity for N-acetylneuraminic acid as well as glycoconjugates containing N-acetylneuraminic acid.     

Studies on the glycopeptides isolated from the urinary protein in heavy-chain disease

The urinary protein excreted in heavy-chain disease was separated by ion-exchange chromatography into two broad fractions designated Cra-1 and Cra-2. For a dimeric molecular weight of approx. 51000, Cra-1 contained 3-4 residues of 6-deoxy-l-galactose (l-fucose), 10 of d-mannose, 5-6 of d-galactose, 12 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine) and 4-5 of N-acetylneuraminic acid (sialic acid), whereas the corresponding values for Cra-2 were 2, 10, 7, 12 and 7. Cra-2 contained in addition 1 residue of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine). Cra-1 contained an average of four oligosaccharide units, two of which contained 1 residue of 6-deoxy-l-galactose, 3 of d-mannose, 1 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, whereas the other two units contained the same proportions of 6-deoxy-l-galactose, d-mannose and 2-acetamido-2-deoxy-d-glucose but 2 residues of d-galactose and 2 of N-acetylneuraminic acid. Cra-2 also contained an average of four oligosaccharide units, but the range of glycopeptides was much wider, containing 0-1 residue of 6-deoxy-l-galactose, 2-3 of d-mannose, 2-3 of d-galactose, 2-3 of 2-acetamido-2-deoxy-d-glucose and 1-3 of N-acetylneuraminic acid. Possible reasons for this heterogeneity are discussed. Glycopeptides were also isolated from Cra-2 that contained 1 residue of d-mannose, 2 of d-galactose, 1 of 2-acetamido-2-deoxy-d-galactose and 0-3 of N-acetylneuraminic acid.     

13C NMR investigation of the anomeric specificity of CMP-N-acetylneuraminic acid synthetase from Escherichia coli.

The anomeric specificity of Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was investigated by NMR using 13C-labeled N-acetylneuraminic acid (NeuAc). Consumption of the beta-anomer of [2-13C]N-acetylneuraminic acid was observed upon addition of enzyme, with a concomitant appearance of an anomeric resonance for CMP-N-acetylneuraminic acid. Inhibition by substrate analogues the anomeric oxygen was determined in a similar manner using [2-13C,(50 atom %)18O]N-acetylneuraminic acid. An upfield shift of 1.5 Hz in the anomeric resonance of both the [13C]NeuAc substrate and CMP-[13C]NeuAc product was observed due to the 18O substitution. This result implies conservation of the NeuAc oxygen. Results of steady-state kinetic analysis suggest a sequential-type mechanism and therefore no covalent intermediate. Thus, CMP-beta-NeuAc is probably formed by a direct transfer of the anomeric oxygen of beta-NeuAc to the alpha-phosphate of CTP.     

Lectin-like binding of Bacillus thuringiensis var. kurstaki lepidopteran-specific toxin is an initial step in insecticidal action

The two delta-endotoxins comprising the Bacillus thuringiensis var. kurstaki HD1 insecticidal protein crystal were separated. The lepidopteran-specific protoxin was activated in vitro and its mechanism of action investigated. Toxicity towards Choristoneura fumiferana CF1 cells was specifically inhibited by preincubation of the toxin with N-acetylgalactosamine and N-acetylneuraminic acid. The lectins soybean agglutinin and wheat germ agglutinin, which bind N-acetylgalactosamine, also inhibited toxicity. Since N-acetylneuraminic acid is not known to occur in insects, these results suggest that the toxin may recognise a specific plasma membrane glycoconjugate receptor with a terminal N-acetylgalactosamine residue.     

Interaction of Trypanosoma cruzi with macrophages: effect of previous incubation of the parasites or the host cells with lectins

The effect of incubation with lectins of the macrophages or two evolutive stages of Trypanosoma cruzi (noninfective epimastigotes and infective trypomastigotes) on the ingestion of the parasites by mouse peritoneal macrophages was studied. Lectins which bind to residues of mannose (Lens culinaris, LCA), N-acetyl-D-glucosamine or N-acetylneuraminic acid (Triticum vulgaris, WGA), beta-D-galactose (Ricinus communis, RCA), N-acetyl-D-galactosamine (Phaseolus vulgaris, PHA; Dolichos biflorus, DBA; and Wistaria floribunda, WFA), fucose (Lotus tetragonolobus, LTA), and N-acetylneuraminic acid (Limulus polyphemus, LPA) were used. By lectin blockage we concluded that, alpha-D-mannose-like, beta-D-galactose and N-acetyl-D-galactosamine (PHA, reagent) residues, located on the macrophage's surface are required for both epi- and trypomastigote uptake, while N-acetylneuraminic acid and fucose residues, impede trypomastigote ingestion but do not interfere with epimastigote interiorization. Macrophages' N-acetyl-D-glucosamine residues are required for epimastigote uptake. On the other hand, from the T. cruzi surface, mannose residues prevent ingestion of epi- and trypomastigotes. Galactose residues participate in endocytosis of trypomastigotes, but hinder epimastigote interiorization. Exposed N-acetyl-D-glucosamine residues are required for uptake of the two evolutive forms. N-acetylneuraminic acid residues on the trypomastigote membrane prevent their endocytosis by macrophages. These results together with those reported previously showing the effect of monosaccharides on the T. cruzi-macrophage interaction, indicate that (a) sugar residues located on the parasite and on macrophage surface play some role in the process of recognition of T. cruzi, (b) different macrophage carbohydrate-containing receptors are involved in the recognition of epimastigotes and trypomastigotes forms of T. cruzi, (c) N-acetylneuraminic acid residues located on the surface of trypomastigotes or macrophages impede the interaction of the parasite with these host cells, and suggest that (d) sugar-binding proteins located on the macrophage surface participate in the recognition of beta-D-galactose and N-acetyl-D-galactosamine residues located on the surface of trypomastigotes and exposed after blockage or splitting off of N-acetylneuraminic acid residues. Some lectins which bind to macrophages and block the ingestion of parasites did not interfere with their adhesion.