Effect of phosphatidylcholine-cholesterol liposomes on Entamoeba histolytica virulence.

Trophozoites of Entamoeba histolytica HM-1:IMSS become less virulent after long-term maintenance in axenic cultures. The factors responsible for the loss of virulence during in vitro cultivation remain unclear. However, it is known that in vitro cultivation of amoeba in culture medium supplemented with cholesterol restores their virulence. In this study, we analyzed the effect of adding phosphatidylcholine-cholesterol (PC-Chol) liposomes to the culture medium and evaluated the effect of this lipid on various biochemical and biological functions of E. histolytica HM-1:IMSS in terms of its virulence. The addition of PC-Chol liposomes to the culture medium maintained the virulence of these parasites against hamster liver at the same level as the original virulent E. histolytica strain, even though these amoebae were maintained without passage through hamster liver for 18 months. The trophozoites also showed increased endocytosis, erythrophagocytosis, and carbohydrate residue expression on the amoebic surface. Protease activities were also modified by the presence of cholesterol in the culture medium. These findings indicate the capacity of cholesterol to preserve amoeba virulence and provide an alternative method for the maintenance of virulent E. histolytica trophozoites without the need for in vivo procedures.

[Chemical and nutritional characteristics from grains of five genotypes of Canavalia ensiformis]. / Características químicas y nutricionales del grano de cinco (5) genotipos de Canavalia ensiformis.

This study evaluated the raw meals from grains of five genotypes of Canavalia ensiformis, by means of the chemical composition, the presence of antinutritional factors (Canavanine and hemaglutination activity) and in vitro protein digestibility. The genotypes studied were: Original, Yaracuy, Tovar, Valle de la Pascua and U-02. The results of chemical composition, showed significance difference between them, except moisture content, found the following average values: Protein 31.37%, fiber: 8.10%, ash: 2.93% and moisture: 11.68%. The canavanine content of the genotypes was variable oscillating between 2.02 and 4.86%, the genotype U-02 presented the higher value, respect to the hemaglutination title changed between: +2 and +5. The in vitro protein digestibility of the raw meals showed significance differences between the genotype, it changed between 47.51% and 51.84%, these values were lower than the casein (97.3%).

[Flour from seeds of Canavalia ensiformis L (DC), raw, stored in an alkaline medium, autoclaved or extracted, in diets for growing pigs]. / Harina de granos de Canavalia ensiformis L (DC) cruda, almacenada en medio alcalino, autoclavada o extruída, en dietas para cerdos en crecimiento.

There were made four nutrition experiments using pork in growing process, with an approximate weight of 14.2 kilograms. In each experiment, it was made a substitution in equals parts of corn flour and soy flour for raw Canavalia flour or processed through alkaline storage, autoclaved or extrusion. In the first three experiments, the substitution level of raw Canavalia (RC), stored in alkaline environment (CAMA) or autoclave (CA) were: 0, 5, 10 and 15%. In the fourth one, the including levels of Canavalia Extruida were: 0, 7.5 and 15%. The raw Canavalia as the Extruida drastically reduced the pork's growth. The Canavalia flour autoclaved (121 degrees C/15 psi/90 min) substantially improved the animal answers, even though the growth in all the substitution levels were lower than the one observed in the original portion. The storage of the beans in alkaline environment, made possible better productive behavior in the animals, and it didn't observe differences (p < 0.01) to increase the witness weight, at the substitution level or 5%. The pork's answer to the Canavalia toxin was manifested in the first term, by a drastic reduction in the voluntary consumption of foods. As a whole, the results indicated that none of the methods used were effective to eliminate or to minimize the toxic effects in the raw Canavalia over the productive behavior of growing pork.

Lectin affinity high-performance liquid chromatography: interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin.

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

Characterization of the endoplasmic reticulum-associated precursor of concanavalin A. Partial amino acid sequence and lectin activity.

Concanavalin A (ConA), which is not a glycoprotein, is synthesized as a glycoprotein precursor (pro-ConA) which is post-translationally processed. This processing results in the loss of a small glycopeptide with a high mannose oligosaccharide. Carrington et al. (Carrington, D.M., Auffret, A., and Hanke, D.E. (1985) Nature 313, 64-66) determined the nucleotide sequence of a cDNA for pro-ConA, and in the derived amino acid sequence the only glycosylation site is in the middle of the molecule. Furthermore, the derived amino acid sequence of the putative precursor of ConA was found not to be colinear with that of ConA. Here we show that pro-ConA is located primarily in an endoplasmic reticulum-rich organelle fraction. Pro-ConA was purified from this fraction and subjected to amino acid sequencing. The first 12 amino acids at the N-terminal end of pro-ConA correspond to amino acids 119-130 of mature ConA, and to amino acids 30-41 of the putative pre-pro-ConA, the sequence of which was derived from the nucleotide sequence of a cDNA. Amino acid sequencing of a tryptic glycopeptide with the high mannose side chain showed that the first 17 amino acids of this peptide correspond to amino acids 154-170 of pre-pro-ConA. The last six amino acids in this series correspond to the first six amino acids of mature ConA. These data fully support the hypothesis of Carrington et al. that the biosynthesis of ConA involves a post-translational peptide cleavage, transposition, and ligation within the original polypeptide. Pro-ConA from the organelle fraction does not bind to Sephadex G-50, indicating that it has no lectin activity. The processing of pro-ConA apparently imparts biological activity to this lectin.

Interaction of concanavalin A with native and denatured forms of jackbean alpha-D-mannosidase.

Tetrameric alpha-D-mannosidase from jackbean is a glycoprotein containing at least one mannosylated oligosaccharide. In the native enzyme, the oligosaccharide is sterically masked from interaction with either endoglucosaminidase H or concanavalin A. Denaturation into subunits permits endoglucosaminidase hydrolysis and removal of the oligosaccharide. The mannosyl residues are attached only to the heavy type of subunit. Removal of the oligosaccharide(s) from the denatured heavy subunit requires the joint action of both alpha-D-mannosidase and N-acetyl-beta-D-glucosaminidase.

Evidence that lectin-binding sites are present on the surface of isolated hypothalamic granules containing luteinizing hormone-releasing hormone.

In the present study, we wished to ascertain if lectin-binding sites are present on hypothalamic granules containing luteinizing hormone-releasing hormone (LHRH). LHRH granules (isolated from homogenates of hypothalami of adult male rats by means of hypo-osmotic shock and differential centrifugation) were subjected to affinity chromatography on columns of concanavalin A (Con A)-Sepharose. The amount of LHRH present in the unbound and bound fraction was measured by radioimmunoassay. Of the total LHRH present in the granule suspension chromatographed, 30 to 40% was bound to the columns, and this binding was not related to the binding of the bulk to the proteins. Synthetic LHRH by itself or synthetic LHRH added to the LHRH granules before chromatography did not bind to Con A-Sepharose, indicating that only LHRH contained within granules is retained on the columns. In addition, we found that the binding of LHRH granules to Con A-Sepharose has a requirement for Ca2+ and Mn2+: after equilibration of Con A-Sepharose with Ca2+ and Mn2+ (prior to the chromatography), all of the LHRH granules were bound to the columns, and this binding was prevented by EDTA. To examine the specificity of the binding of LHRH granules to Con A-Sepharose, a competing sugar (alpha-methyl-D-mannoside (alpha-MM)) was added to the LHRH granules, and the columns were equilibrated with alpha-MM prior to chromatography. Under this condition, the Ca2+- and Mn2+-dependent binding of LHRH granules to Con A-Sepharose was inhibited. In addition to Con A-Sepharose, we observed that LHRH granules bind to wheat germ agglutinin-Sepharose but not to Sepharose which does not contain a lectin. These findings are indicative that lectin-binding sites, carbohydrate in nature, are present on the surface of isolated LHRH granules. We propose that such binding sites may play a role in the release of LHRH from hypothalamic neurons.

Structure of soncanavalin A at 4 A resolution.

Concanavalin A, a phytohemagglutinin isolated from the jack bean, crystallizes at pH 6.8 in the orthorhombic space group 1222 with a = 89.9, b = 87.2, and c = 63.1 A. We have analyzed x-ray diffraction intensity data to 4 A resolution on native concanavalin A and five heavy-metal derivatives: lead, mersalyl, chloroplatinate, uranyl, and o-mercuri-p-nitrophenol. Heavy-atom positions, occupancies, and isotropic thermal parameters have been refined by least-squares methods. The electron density maps clearly show the molecular shape and the packing of the concanavalin A molecules. The asymmetric unit (mol wt 27,000) forms an elliptical dome or "gumdrop" with a base of approximately 46 x 26 A and a height of 42 A. The subunits are paired across 2-fold axes parallel to the c-axis to form dimers. The dimers are in turn paired across points of D(2) symmetry to form tetramers of roughly tetrahedral shape. Each unit has a depression located on the surface which could be the site of saccharide binding. In many regions we have been able to trace the course of the polypeptide chain.