Topography of the combining region of a Thomsen-Friedenreich-antigen-specific lectin jacalin (Artocarpus integrifolia agglutinin). A thermodynamic and circular-dichroism spectroscopic study

Thermodynamic analysis of carbohydrate binding by Artocarpus integrifolia (jackfruit) agglutinin (jacalin) shows that, among monosaccharides, Me alpha GalNAc (methyl-alpha-N-acetylgalactosamine) is the strongest binding ligand. Despite its strong affinity for Me alpha GalNAc and Me alpha Gal, the lectin binds very poorly when Gal and GalNAc are in alpha-linkage with other sugars such as in A- and B-blood-group trisaccharides, Gal alpha 1-3Gal and Gal alpha 1-4Gal. These binding properties are explained by considering the thermodynamic parameters in conjunction with the minimum energy conformations of these sugars. It binds to Gal beta 1-3GalNAc alpha Me with 2800-fold stronger affinity over Gal beta 1-3GalNAc beta Me. It does not bind to asialo-GM1 (monosialoganglioside) oligosaccharide. Moreover, it binds to Gal beta 1-3GalNAc alpha Ser, the authentic T (Thomsen-Friedenreich)-antigen, with about 2.5-fold greater affinity as compared with Gal beta 1-3GalNAc. Asialoglycophorin A was found to be about 169,333 times stronger an inhibitor than Gal beta 1-3GalNAc. The present study thus reveals the exquisite specificity of A. integrifolia lectin for the T-antigen. Appreciable binding of disaccharides Glc beta 1-3GalNAc and GlcNAc beta 1-3Gal and the very poor binding of beta-linked disaccharides, which instead of Gal and GalNAc contain other sugars at the reducing end, underscore the important contribution made by Gal and GalNAc at the reducing end for recognition by the lectin. The ligand-structure-dependent alterations of the c.d. spectrum in the tertiary structural region of the protein allows the placement of various sugar units in the combining region of the lectin. These studies suggest that the primary subsite (subsite A) can accommodate only Gal or GalNAc or alpha-linked Gal or GalNAc, whereas the secondary subsite (subsite B) can associate either with GalNAc beta Me or Gal beta Me. Considering these factors a likely arrangement for various disaccharides in the binding site of the lectin is proposed. Its exquisite specificity for the authentic T-antigen, Gal beta 1-3GalNAc alpha Ser, together with its virtual non-binding to A- and B-blood-group antigens, Gal beta 1-3GalNAc beta Me and asialo-GM1 should make A. integrifolia lectin a valuable probe for monitoring the expression of T-antigen on cell surfaces.

Localized agglutinin staining in muscle capillaries from normal and very old atrophic human muscle using winged bean ( Psophocarpus tetragonolobus ) lectin

The winged bean (Psophocarpus tetragonolobus) agglutinin (total lectin) and its basic (WBA I) and acidic isoform (WBA II) were used to analyze capillaries in sections from human muscle. The microvessels were clearly labeled after incubation with the lectins in both normal muscle and in old muscles with age-related type II atrophy or muscle fiber grouping. Muscle fibers, nerves, and connective tissue remained unstained. The total lectin detected muscle capillaries from all blood group AB0 individuals. The isoform WBA I reacted only with blood vessels in blood group A and B individuals, while the blood vessels in blood group 0 individuals were demonstrated with WBA II. WBA I staining was inhibited by p-nitrophenyl α-galactopyranoside and N-acetylgalactosamine, whereas 2′-fucosyllactose and preincubation with an antibody against type-1 chain H abolished capillary staining with WBA II. The study demonstrates the usefulness of WBA as a marker of capillaries in human muscle.

Carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus)

The carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus) was investigated by quantitative precipitin analysis using blood group A, B, H, Le and I substances and by precipitation inhibition with various mono- and oligosaccharides. The lectin precipitated best with A1 substances and moderately with B and A2 substances, but not with H or Le substances. Inhibition assays of lectin-blood group A1 precipitation demonstration that A substance-derived oligosaccharides having the common structure: d-Ga1NAcα(1 → 3)d-Gal-(β1 → Image ) to a d-Glc, were the best inhibitors and about 8 and 4 times more active than d-Ga1NAc and d-Ga1NAcα(1 → 3)d-Ga1, respectively. A difucosyl A-specific oligosaccharide (A-penta), a monofucosyl (A-tetra) and a non-fucosyl containing (A5 II) oligosaccharide, d-Ga1NAcα(1 → 3)d-Ga1β(1 → 3)d-G1cNAc, had almost the same reactivity, suggesting that the fucose linked to the sub-terminal d-Ga1 or to the third sugar, d-GlcNAc, from the non-reducing end made no contribution to the carbohydrate binding. Although a terminal non-reducing d-Ga1NAc or d-Ga1 residue was indispensible for binding, the lectin bound not only to these terminal non-reducing galactopyranosyl residues, but also showed increased binding to oligosaccharides in which it was bonded to a sub-terminal d-Ga1 joined to a d-GlcNAc residue, as in blood group A or B substances. This defines the site, thus far, as complementary to a disaccharide plus the β linkage to the third sugar (d-Glc or d-GlcNAc) from the non-reducing end. The role of the β(1 → 3) or β(1 → 4) linkage of the sub-terminal non-reducing d-Gal to the d-GlcNAc requires further study.

Erythrocyte-binding studies on an acidic lectin from winged bean (Psophocarpus tetragonolobus)

n acidic lectin (WBA II) was isolated to homogeneity from the crude seed extract of the winged bean (Psophocarpus tetragonolobus) by affinity chromatography on lactosylaminoethyl-Bio-Gel. Binding of WBA II to human erythrocytes of type-A, -B and -O blood groups showed the presence of 10(5) receptors/cell, with high association constants (10(6)-10(8) M-1). Competitive binding studies with blood-group-specific lectins reveal that WBA II binds to H- and T-antigenic determinants on human erythrocytes. Affinity-chromatographic studies using A-, B-, H- and T-antigenic determinants coupled to an insoluble matrix confirm the specificity of WBA II towards H- and T-antigenic determinants. Inhibition of the binding of WBA II by various sugars show that N-acetylgalactosamine and T-antigenic disaccharide (Thomsen-Friedenreich antigen, Gal beta 1-3GalNAc) are the most potent mono- and di-saccharide inhibitors respectively. In addition, inhibition of the binding of WBA II to erythrocytes by dog intestine H-fucolipid prove that the lectin binds to H-antigenic determinant.

Thermodynamics of lectin-sugar interaction: Binding of sugars to winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBAI)

Combining site of WBAI is extended and encompasses all the residues of blood group A-reactive trisaccharide [GalNAcalpha3Galbeta4Glc]. Though both of the fucose residues of A-pentasaccharide [GalNAcalpha(Fucalpha2)3Galbeta(Fucalpha3)4Glc] do not directly interact, with the combining site they thermodynamically favour the interaction of GalNAcalpha3Galbeta4Glc part of the molecule by imposing a sterically favourable orientation of the binding epitope viz. GalNAcalpha3Galbeta4Glc of the saccharide. Binding of sugars is driven by enthalpy and is devoid of heat capacity changes. This together with enthalpy-entropy compensation observed for these processes underscore the importance of water reorganization as being one of the principal determinant of protein-sugar interactions.

Human alveolar T lymphocyte responses to Mycobacterium tuberculosis antigens: role for CD4+ and CD8+ cytotoxic T cells and relative resistance of alveolar macrophages to lysis.

T cell-mediated cytotoxicity against Mycobacterium tuberculosis (MTB)-infected macrophages may be a major mechanism of specific host defense, but little is known about such activities in the lung. Thus, the capacity of alveolar lymphocyte MTB-specific cell lines (AL) and alveolar macrophages (AM) from tuberculin skin test-positive healthy subjects to serve as CTL and target cells, respectively, in response to MTB (H37Ra) or purified protein derivative (PPD) was investigated. Mycobacterial Ag-pulsed AM were targets of blood CTL activity at E:T ratios of > or = 30:1 (51Cr release assay), but were significantly more resistant to cytotoxicity than autologous blood monocytes. PPD- plus IL-2-expanded AL and blood lymphocytes were cytotoxic for autologous mycobacterium-stimulated monocytes at E:T ratios of > or = 10:1. The CTL activity of lymphocytes expanded with PPD was predominantly class II MHC restricted, whereas the CTL activity of lymphocytes expanded with PPD plus IL-2 was both class I and class II MHC restricted. Both CD4+ and CD8+ T cells were enriched in BL and AL expanded with PPD and IL-2, and both subsets had mycobacterium-specific CTL activity. Such novel cytotoxic responses by CD4+ and CD8+ T cells may be a major mechanism of defense against MTB at the site of disease activity.

Ethnobotany of the Monpa ethnic group at Arunachal Pradesh, India

Background: The present paper documents the uses of plants in traditional herbal medicine for human and veterinary ailments, and those used for dietary supplements, religious purpose, local beverage, and plants used to poison fish and wild animals. Traditional botanical medicine is the primary mode of healthcare for most of the rural population in Arunachal Pradesh. Materials and methods: Field research was conducted between April 2006 and March 2009 with randomly selected 124 key informants using semi-structured questionnaire. The data obtained was analyzed through informant consensus factor (F(IC)) to determine the homogeneity of informant's knowledge on medicinal plants. Results: We documented 50 plants species belonging to 29 families used for treating 22 human and 4 veterinary ailments. Of the medicinal plants reported, the most common growth form was herbs (40%) followed by shrubs, trees, and climbers. Leaves were most frequently used plant parts. The consensus analysis revealed that the dermatological ailments have the highest F(IC) (0.56) and the gastro-intestinal diseases have F(IC) (0.43). F(IC) values indicated that there was high agreement in the use of plants in dermatological and gastro-intestinal ailments category among the users. Gymnocladus assamicus is a critically rare and endangered species used as disinfectant for cleaning wounds and parasites like leeches and lice on livestocks. Two plant species (Illicium griffithii and Rubia cordifolia) are commonly used for traditional dyeing of clothes and food items. Some of the edible plants recorded in this study were known for their treatment against high blood pressure (Clerodendron colebrookianum), diabetes mellitus (Momordica charantia), and intestinal parasitic worms like round and tape worms (Lindera neesiana, Solanum etiopicum, and Solanum indicum). The Monpas of Arunachal Pradesh have traditionally been using Daphne papyracea for preparing hand-made paper for painting and writing religious scripts in Buddhist monasteries. Three plant species (Derris scandens, Aesculus assamica, and Polygonum hydropiper) were frequently used to poison fish during the month of June-July every year and the underground tuber of Aconitum ferrox is widely used in arrow poisoning to kill ferocious animals like bear, wild pigs, gaur and deer. The most frequently cited plant species; Buddleja asiatica and Hedyotis scandens were used as common growth supplements during the preparation of fermentation starter cultures. Conclusion: The traditional pharmacopoeia of the Monpa ethnic group incorporates a myriad of diverse botanical flora. Traditional knowledge of the remedies is passed down through oral traditions without any written document. This traditional knowledge is however, currently threatened mainly due to acculturation and deforestation due to continuing traditional shifting cultivation. This study reveals that the rural populations in Arunachal Pradesh have a rich knowledge of forest-based natural resources and consumption of wild edible plants is still an integral part of their socio-cultural life. Findings of this documentation study can be used as an ethnopharmacological basis for selecting plants for future phytochemical and pharmaceutical studies.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-gamma production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-alpha and IFN-gamma) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-gamma and IL-17 production in blood from TB plus patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras

Background: A better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates. Methods: Recombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-gamma and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29). Results: M.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-gamma production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c. Conclusions: The pattern of immune target recognition is different in regard to IFN-gamma and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.