ABSTRACT
AIM OF THE STUDY: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. It comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). MATERIAL AND METHODS: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII) and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. RESULTS: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. CONCLUSIONS: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production.
Subject(s)
Calotropis/chemistry , Latex/pharmacology , Leukocyte Rolling/drug effects , Nitric Oxide/immunology , Peritonitis/immunology , Plant Proteins/immunology , Animals , Cell Adhesion/drug effects , Cell Movement/drug effects , Male , Mesentery/blood supply , Nitric Oxide/blood , Peritonitis/chemically induced , Rats , Rats, WistarABSTRACT
A simple and inexpensive method was developed to rapidly define the specificity of mannose-specific lectins toward oligomannoside-type structures. The method involved the interaction of a mixture of N-[(14)C]-acetylated glycoasparagines, prepared by exhaustive pronase digestion of bovine pancreatic ribonuclease B and N-[(14)C]-acetylation with [(14)C]-acetic anhydride and containing all the possible oligomannoside-type N-glycans, with the lectin immobilized on Sepharose-4B. After exhaustive desalting, the obtained fractions were separated by high-performance thin-layer chromatography on silica gel plates and visualized by autoradiography with intensifying screen. As an example of the usefulness of this method, the fine specificity of artocarpin, the mannose-specificity lectin isolated from seeds of jackfruit (Artocarpus integrifolia) toward oligomannoside-type structures is presented. On the basis of such a determination, the best oligomannosidic ligand recognized by a mannose-specific lectin can be selected for studies of crystal structures of the lectin in complex with the defined ligand. Furthermore, some of these immobilized lectins, after definition of their precise specificities with the method, could represent valuable tools for the fractionation and characterization of oligomannose-type structures, present in complex mixtures.
Subject(s)
Mannose-Binding Lectins/metabolism , Microchemistry/methods , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Animals , Artocarpus , Carbohydrate Sequence , Cattle , Chemical Fractionation , Chromatography, Affinity , Chromatography, Agarose , Chromatography, High Pressure Liquid/methods , Glycopeptides/chemistry , Glycopeptides/metabolism , Mannose-Binding Lectins/chemistry , Mannose-Binding Lectins/isolation & purification , Molecular Sequence Data , Oligosaccharides/isolation & purification , Plant Lectins/chemistry , Plant Lectins/isolation & purification , Plant Lectins/metabolism , Ribonucleases/chemistry , Sensitivity and Specificity , Substrate SpecificityABSTRACT
The embryotoxic activity and differential binding of plant-derived carbohydrate-recognizing proteins on sea urchin (Lytechinus variegatus) embryo cells was investigated. IC50 doses for toxicity on larvae development varied from 0.6 up to 96.3 microg ml(-1) and these effects were largely reversed by previously heating the proteins. Changes in the glycoconjungate status of the cell surface were assessed by time-course binding of the proteins during embryogenesis according to their carbohydrate-binding specificity. Glucose/mannose binding-proteins bound embryo cells at the same stage of development, at a similar stage to the N-acetylglucosamine/N-acetylneuraminic acid binding-protein (WGA) and earlier than galactose specific ones. FITC-conjugates of these proteins confirmed the above results and revealed the presence of specific and differential receptors for them. Inhibition assays using inhibitory glycoproteins significantly diminished the labelled patterns of FITC-conjugates. In conclusion, the assayed proteins exhibited embryotoxicity and their binding requirements were useful for following changes in the pattern of cell surface glycoconjugates on embryo cells of sea urchin. This property could be useful in analyzing other cell types.
