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Abstract: In this study, we tested the potential use of a lectin from Lonchocarpus sericeus seeds (LSL), to control neutrophil migration and inflammatory hypernociception (decrease of nociceptive threshold). Pretreatment of the animals intravenously (15 min before) with LSL inhibited neutrophil migration to the peritoneal cavity in a dose-dependent fashion confirmed by an inhibition of rolling and adhesion of leukocytes by intravital microscopy. We also tested the ability of the pretreatment with LSL to inhibit neutrophil migration on immunised mice, and it was observed that a strong inhibition of neutrophil migration induced by ovoalbumin in immunized mice. Another set of experiments showed that pretreatment of the animals with LSL, inhibited the mechanical hypernociception in mice induced by the i.pl. injection of OVA in immunized mice and of carrageenan in naïve mice, but not that induced by prostaglandin E2 (PGE2) or formalin. This anti-nociceptive effect correlated with an effective blockade of neutrophil influx, as assessed by the hind paw tissue myeloperoxidase levels. In addition, we measured cytokines (TNF-α and IL-1β) and chemokines (MIP-1α [CCL3] and KC [CXCL1]) from the peritoneal exudates and i.pl. tissue. Animals treated with LSL showed inhibition of cytokines and chemokines release in a dose-dependent manner. In conclusion, we demonstrated that the inhibitory effects of LSL on neutrophil migration and mechanical inflammatory hypernocicepetion are associated with the inhibition of the production of cytokines and chemokines. [Copyright &y& Elsevier]

Isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements demonstrate that a chemically and enzymatically prepared form of porcine submaxillary mucin that possesses a molecular mass of ~106 daltons and ~2300 α-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a Kd of 0.2 nM, which is ~106-fold enhanced affinity relative to GalNAcα1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing ~23 α-GalNAc residues binds with ~103-fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing ~11-12 α-GalNAc residues shows ~102-fold enhanced affinity. A natural carbohydrate decorated form of PSM (Fd-PSM) containing 40% of the core 1 blood group type A tetrasaccharide, and 58% peptide-linked GalNAcα1-O-Ser/Thr residues, with 45% of the peptide-linked α-GalNAc residues linked α-(2,6) to N-glycolylneuraminic acid, shows ~104 enhanced affinity for SBA. Vatairea macrocarpa lectin (VML), which is also a GalNAc binding lectin, displays a similar pattern of binding to the four forms of PSM, although there are quantitative differences in its affinities as compared with SBA. The higher affinities of SBA and VML for Tn-PSM relative to Fd-PSM indicate the importance of carbohydrate composition and epitope density of mucins on their affinities for lectins. The higher affinities of SBA and VML for Tn-PSM relative to its two shorter chain analogs demonstrate that the length of a mucin polypeptide and hence total carbohydrate valence determines the affinities of the three Tn-PSM analogs. The results suggest a binding model in which lectin molecules ‘bind and jump’ from α-GalNAc residue to α-GalNAc residue along the polypeptide chain of Tn-PSM before dissociating. The complete thermodynamic binding parameters for these mucins including their binding stoichiometries are presented. The results have important implications for the biological activities of mucins including those expressing the Tn cancer antigen. [ABSTRACT FROM AUTHOR]

Lectins from plants of the Diocleinae subtribe often exhibit specificity towards mannose/glucose and derived sugars, with some plants also displaying a second lectin specific to lactose/GalNAc. Here, we present a novel lectin from Collaea speciosa , named CsL, that displays specificity for GlcNAc/glucose. The lectin was extracted from Collaea speciosa seeds and purified by a single chromatographic step on a Sephadex G-50 matrix. In solution, the lectin appears as a dimeric protein composed of 25 kDa monomers. The protein is stable at pH 7–8 and dependent on divalent cations. CsL maintained its agglutination activity after heating to 90 °C for 1 h. Glycan array studies revealed that CsL binds to N -glycans with terminal GlcNAc residues, chitobiose and chitotriose moieties. The partial amino acid sequence of the lectin is similar to that of some lactose-specific lectins from the same subtribe. In contrast to other ConA-like lectins, CsL is not toxic to Artemia. Because of its remarkably different properties and specificity, this lectin could be the first member of a new group inside the Diocleinae lectins. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme is the most aggressive type of glioma, with limited treatment and poor prognosis. Despite some advances over the last decade, validation of novel and selective antiglioma agents remains a challenge in clinical pharmacology. Prior studies have shown that leguminous lectins may exert various biological effects, including antitumor properties. Accordingly, this study aimed to evaluate the mechanisms underlying the antiglioma activity of ConBr, a lectin extracted from the Canavalia brasiliensis seeds. ConBr at lower concentrations inhibited C6 glioma cell migration while higher levels promoted cell death dependent upon carbohydrate recognition domain (CRD) structure. ConBr increased p38MAPK and JNK and decreased ERK1/2 and Akt phosphorylation. Moreover, ConBr inhibited mTORC1 phosphorylation associated with accumulation of autophagic markers, such as acidic vacuoles and LC3 cleavage. Inhibition of early steps of autophagy with 3-methyl-adenine (3-MA) partially protected whereas the later autophagy inhibitor Chloroquine (CQ) had no protective effect upon ConBr cytotoxicity. ConBr also augmented caspase-3 activation without affecting mitochondrial function. Noteworthy, the caspase-8 inhibitor IETF-fmk attenuated ConBr induced autophagy and C6 glioma cell death. Finally, ConBr did not show cytotoxicity against primary astrocytes, suggesting a selective antiglioma activity. In summary, our results indicate that ConBr requires functional CRD lectin domain to exert antiglioma activity, and its cytotoxicity is associated with MAPKs and Akt pathways modulation and autophagy- and caspase-8- dependent cell death. • ConBr lectin inhibits cell migration and promotes C6 glioma cell death by autophagy. • ConBr-induced cell death and autophagy depends upon caspase-8 activity. • ConBr stimulates p38MAPK and JNK and inhibits ERK, Akt and mTORC1 in glioma cells. • ConBr displays selective cytotoxicity against glioma cells as compared to astrocytes. [ABSTRACT FROM AUTHOR]

Lectins from Diocleinae subtribe species (family Leguminosae) are of special interest since they present a wide spectrum of biological activities, despite their high structural similarity. During their synthesis in plant cells, these proteins undergo post-translational processing resulting in the formation of three chains (α, β, γ), which constitute the lectins' subunits. Furthermore, such wild-type proteins are presented as isolectins or with different combinations of these chains, which undermine their biotechnological potential. Thus, the present study aimed to produce a recombinant form of the lectin from Dioclea sclerocarpa seeds (DSL), exclusively constituted by α-chain. The recombinant DSL (rDSL) was successfully expressed in E. coli BL21 (DE3) and purified by affinity chromatography (Sephadex G-50), showing a final yield of 74 mg of protein per liter of culture medium and specificity for D-mannose, α-methyl-mannoside and melibiose, unlike the wild-type protein. rDSL presented an effective vasorelaxant effect in rat aortas up to 100% and also interacted with glioma cells C6 and U87. Our results demonstrated an efficient recombinant production of rDSL in a bacterial system that retained some biochemical properties of the wild-type protein, showing wider versatility in sugar specificities and better efficacy in its activity in the biological models evaluated in this work. [ABSTRACT FROM AUTHOR]

The antitumor activity of DVL, a lectin purified from Dioclea violacea seeds, on the U87 human glioma cell line was evaluated and compared with Canavalia ensiformis lectin (ConA). Treatment with DVL (10–100 μg/mL; 24–96 h) induced alterations in cell morphology, decreased cell numbers and clonogenic survival in a time- and concentration-dependent manner. DVL caused significant decreases in cell viability and impaired cell migration. Mechanistically, DVL treatment (12 h) disrupted mitochondrial electrochemical gradient, without ROS accumulation or caspase activation. In the absence of apoptosis, DVL (30–100 μg/mL), instead, induced autophagy, as detected by acridine orange staining and cleavage of LC3I. Inhibition of autophagy with 3-Methyladenine (3-MA) and Chloroquine partially abrogated DVL, but not ConA, cytotoxicity. The modulation of signaling pathways that orchestrate autophagic and cell survival processes were analyzed. DVL (30–100 μg/mL) decreased Akt, mTORC1 and ERK1/2 phosphorylation and augmented JNK(p54) and p38MAPK phosphorylation. DVL was more potent than ConA for most parameters analyzed. Even though both lectins showed cytotoxicity to glioma cells, they spared primary astrocyte cultures. The results suggest a selective antiglioma activity of DVL by inhibiting U87 glioma cell migration and proliferation and inducing cell death, partially associated with autophagy, and likely involving Akt and mTORC1 dephosphorylation. • Dioclea violacea lectin (DVL) caused significant decrease of U87 glioma cell viability and impaired cell migration. • DVL disrupted mitochondrial electrochemical gradient, without ROS accumulation or caspase activation. • DVL induced autophagy in U87 glioma cells by a mechanism likely involving Akt and mTORC1 inhibition. • Antiglioma activity of DVL was more potent than ConA. [ABSTRACT FROM AUTHOR]

Abstract Inflammation of temporomandibular joint (TMJ) tissues are the most common cause of pain conditions associated with temporomandibular disorders (TMDs). After a tissue and/or neural damage, the inflammatory response is characterized by plasma extravasation and leukocytes infiltration in the TMJ tissues, which in turn, release inflammatory cytokines cascades responsible for inflammatory pain. Lectins are glycoproteins widely distributed in nature that may exhibit anti-inflammatory properties. This study demonstrated by molecular docking and MM/PBSA that the lectin from Dioclea violacea (DVL) interacts favorably with α-methyl-D-mannoside, N- acetyl-D-glucosamine, and core1-sialyl-Lewis X which are associated with leukocytes migration during an inflammatory response. Wistar rats pretreated with intravenously injection of DVL demonstrated a significant inhibition of plasma extravasation induced by carrageenan (a non-neurogenic inflammatory inductor) and mustard oil (a neurogenic inflammatory inductor) in the TMJ periarticular tissues (p < 0.05; ANOVA, Tukey's test). In addition, DVL significantly reduced carrageenan-induced leukocyte migration in the TMJ periarticular tissues mediated by down-regulation of ICAM-1 expression. These results suggest a potential anti-inflammatory effect of DVL in inflammatory conditions of TMJ. Highlights • DVL reduces carrageenan- and mustard oil-induced TMJ inflammation. • DVL reduces the ICAM-1 expression in TMJ periarticular tissue. • DVL may act as anti-inflammatory agent in TMJ disorders. [ABSTRACT FROM AUTHOR]

Abstract Plant lectins have been studied owing to their structural properties and biological effects that include agglutinating activity, antidepressant-like effect and antitumor property. The results from this work showed the effects of the lectin extracted from the Dioclea violacea plant (DVL) on the C6 rat glioma cell line. DVL treatment was able to induce caspase-3 activation, apoptotic cell death and cellular membrane damage. Furthermore, DVL decreased mitochondrial membrane potential and increased the number of acidic vesicles and cleavage of LC3, indicating activation of autophagic processes. DVL also significantly inhibited cell migration. Compared to ConA, a well-studied lectin extracted from Canavalia ensiformes seeds, some effects of DVL were more potent, including decreasing C6 glioma cell viability and migration ability. Taken together, the results suggest that DVL can induce glioma cell death, autophagy and inhibition of cell migration, displaying potential anti-glioma activity. [ABSTRACT FROM AUTHOR]

With important carbohydrate binding properties, lectins are proteins able to decipher the glycocode, and as such, they can be used in bioassays involving cell–cell communication, protein targeting, inflammation, and hypernociception, among others. In this study, a new glucose/mannose-specific lectin from Canavalia villosa seeds (Cvill) was isolated by a single affinity chromatography step in a Sephadex ® G-50 column, with a purification yield of 19.35 mg of lectin per gram of powdered seed. Analysis of intact protein by mass spectrometry showed the lectin is composed of three polypeptide chains, including a 25.6 kDa α chain, 12.9 KDa β, and 12.6 KDa γ fragments, similar to the profile of ConA-like glucose/mannose-specific lectins. Partial sequence of the protein was obtained by MS-MALDI TOF/TOF covering 41.7% of its primary structure. Cvill presented sugar specificity to d -glucose, α-methyl- d -mannoside, d -mannose, and glycoproteins fetuin and ovoalbumin. The lectin characterization showed that Cvill presents high stability within a broad range of pH and temperature, also showing average toxicity against Artemia nauplii . The proinflammatory effect of Cvill was observed by induction of paw edema and hypernociception in mice, with the participation of the carbohydrate binding site, showing its potential to be used as tool in inflammation studies. [ABSTRACT FROM AUTHOR]

The lectin DLasiL was isolated from seeds of the Dioclea lasiocarpa collected from the northeast coast of Brazil and characterized for the first time by mass spectrometry, DNA sequencing, inductively coupled plasma-mass spectrometry, electron paramagnetic resonance, and fluorescence spectroscopy. The structure of DLasiL lectin obtained by homology modelling suggested strong conservation of the dinuclear Ca/Mn and sugar-binding sites, and dependence of the solvent accessibility of tryptophan-88 on the oligomerisation state of the protein. DLasiL showed highly potent (low nanomolar) antiproliferative activity against several human carcinoma cell lines including A2780 (ovarian), A549 (lung), MCF-7 (breast) and PC3 (prostate), and was as, or more, potent than the lectins ConBr ( Canavalia brasiliensis ), ConM ( Canavalia maritima ) and DSclerL ( Dioclea sclerocarpa ) against A2780 and PC3 cells. Interestingly, DLasiL lectin caused a G2/M arrest in A2780 cells after 24 h exposure, activating caspase 9 and delaying the on-set of apoptosis. Confocal microscopy showed that fluorescently-labelled DLasiL localized around the nuclei of A2780 cells at lectin doses of 0.5–2 × IC 50 and gave rise to enlarged nuclei and spreading of the cells at high doses. These data reveal the interesting antiproliferative activity of DLasiL lectin, and suggest that further investigations to explore the potential of DLasiL as a new anticancer agent are warranted. [ABSTRACT FROM AUTHOR]

Vatairea guianensis lectin (VGL), Dalbergiae tribe, is a N-acetyl-galactosamine (GalNAc)/Galactose (Gal) lectin previously purified and characterized. In this work, we report its structural features, obtained from bioinformatics tools, and its inflammatory effect, obtained from a rat paw edema model. The VGL model was obtained by homology with the lectin of Vatairea macrocarpa (VML) as template, and we used it to demonstrate the common characteristics of legume lectins, such as the jellyroll motif and presence of a metal-binding site in the vicinity of the carbohydrate-recognition domain (CRD). Protein-ligand docking revealed favorable interactions with N- acetyl- d -galactosamine, d -galactose and related sugars as well as several biologically relevant N- and O -glycans. In vivo testing of paw edema revealed that VGL induces edematogenic effect involving prostaglandins, interleukins and VGL CRD. Taken together, these data corroborate with previous reports showing that VGL interacts with N- and/or O- glycans of molecular targets, particularly in those presenting galactosides in their structure, contributing to the lectin inflammatory effect. [ABSTRACT FROM AUTHOR]

We report the successful fabrication of a novel gold surface coated with a thiol-lectin multilayer that showed specific interaction towards D-mannose sugars. Firstly, the gold surface was spontaneously modified with gliotoxin (gtx), a sulfur containing diketopiperazine isolated from marine-derived fungus, and fully characterized by electrochemical, EIS, SPR and SERS techniques. The results indicated the sulfur bridge of gtx is broken upon adsorption and the molecules are flat lying on gold with surface coverage of 2.3 × 10 −10 mol cm −2 and reductive desorption potential of −0.87 V vs Ag/AgCl, i.e. strongly chemisorbed on gold. The real-time monitoring of the immobilization of Concanavalin Br (ConBr) lectin (6.9 × 10 −12 mol cm −2 ) on the gold surface modified with gliotoxin (Au/gtx) was performed by SPR which was also used to prove the recognition capability of ConBr towards D-mannose remains active on Au/gtx. Comparative studies using 11-mercaptoundecanoic acid and cysteine on gold show the interaction between the thiol layer with ConBr involves a network of hydrogen bonds and that the SAM formed with gliotoxin is the most robust monolayer. Impedimetric measurements in solution containing [Fe(CN) 6 ] 3−/4− redox probe and D-mannose sugar at different concentrations indicate hydrogen bonds are the major intermolecular contributions connecting the sugar molecules to the sugar recognition sites of ConBr where Ca 2+ and Mn 2+ ions are inserted.. [ABSTRACT FROM AUTHOR]

Legume lectins are the most thoroughly studied group of lectins and have been widely linked to many pathological processes. Their use as immunohistochemistry markers for cell profiling and cancer diagnosis have made these molecules important tools for immunological studies and have stimulated the prospection and characterization of new lectins. The crystal structures of a recombinant seed lectin from Vatairea macrocarpa (rVML) and its complexes with GalNAcα1-O-Ser, GalNAc and α-lactose, have been determined at 1.90, 1.97, 2.70 and 1.83 Å resolution, respectively. Small angle X-ray scattering and calorimetry assays have confirmed the same pH stable oligomerization pattern and binding profiles proposed for its wild-type counterpart. In silico analyzes have explored the potential of this recombinant lectin as new tool for cancer research through a comparative profile with other legume lectins widely used for cancer diagnosis and prognosis. The results suggest the recognition of specific epitopes exhibited on different cancer cells as a process that relies on the disposition of hydrophobic clusters and charged regions around the lectin carbohydrate-binding site, favouring the anchorage of different groups in the antigen boundaries, highlighting the different potential of each analyzed lectin. In conclusion, the experimental results and comparative analysis show that rVML is as a promising tool for cancer research, able to bind with high affinity specific tumor-associated antigens, highly stable and easily produced. [ABSTRACT FROM AUTHOR]

Lectins are proteins that show a variety of biological activities. However, they share in common at least one domain capable of recognizing specific carbohydrates reversibly without changing its structure. The legume lectins family is the most studied family of plant lectins, in particular the Diocleinae subtribe, which possesses high degree of structural similarity, but variable biological activities. This variability lies in small differences that can be analyzed in studies based on structures. In particular, Dioclea sclerocarpa seed lectin (DSL) presents low ability to relax endothelialized rat aorta in comparison with other Dioclea lectins such as Dioclea violacea (DVL), Dioclea virgata (DvirL) and Dioclea rostrata (DRL). The DSL relaxation mechanism relies on nitric oxide production and carbohydrate recognition domain (CRD). This feature can be explained by structural differences, since DSL has a carbohydrate recognition domain design less favorable. In addition, the presence of a glutamate residue at position 205 proved to be a decisive factor for the low relaxant effect of Dioclea lectins. [ABSTRACT FROM AUTHOR]

Resveratrol can also inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro- and anti-inflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5 mM for 2 h of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an R factor of 16.0% and an R free of 25.5%. Resveratrol binds in the rigid β-sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth β-strands of the rigid β-sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of the compound 1,4-dihydroxy-2E,6E,12E-trien-5-one-casbane (CD1CN), a casbane diterpene isolated from Croton nepetaefolius stalks, on the biofilm formation or preformed biofilms of bacteria and yeasts. Minimum inhibitory concentration results showed that CD1CN inhibited the growth of single cultures of Staphylococcus aureus and Candida albicans at 125 and 500 μg/mL, respectively, as well as dual cultures of S. aureus with C. albicans or Candida glabrata at 500 and 250 μg/mL, respectively. In general, CD1CN reduced biofilm biomass when applied to preformed biofilms or when applied during the biofilm formation of single and dual cultures in concentrations ranging from 31.25 to 250 μg/mL, depending on the culture. CD1CN was more effective in reducing the cfu of S. aureus in single and dual biofilms (62.5–250 μg/mL) than that of Pseudomonas aeruginosa , although this reduction was also significant. For yeasts, CD1CN was generally more effective in reducing C. glabrata cfu in single or dual cultures when compared to C. albicans . SEM images of the dual-species biofilms confirmed these results. In conclusion, CD1CN could be an effective alternative to conventional antimicrobial agents against infectious biofilms, in particular those attributed to mixed cultures. [ABSTRACT FROM AUTHOR]

Abstract: Lectins recognize and reversibly bind to carbohydrates attached to proteins and lipids modulating a variety of signaling pathways. We previously showed that ConBr, a lectin from Canavalia brasiliensis seeds, produced an antidepressant-like effect in mice by modulating the monoaminergic neurotransmitter systems. Moreover, ConBr blocked hippocampal neurotoxicity induced by quinolinic acid in vivo and by glutamate in vitro, suggesting a neuroprotective activity of ConBr via glutamatergic system modulation. Therefore, the present study was undertaken to investigate the involvement of the N-methyl-d-aspartate (NMDA) receptor and the l-arginine–nitric oxide (NO) pathway in the antidepressant-like action displayed by ConBr in the forced swimming test (FST). With the aim of verifying the involvement of NMDA receptors in the antidepressant-like effect of ConBr (10μg/site, i.c.v.), an intracerebroventricular (i.c.v.) pretreatment with either NMDA (0.1pmol/site) or d-serine (30μg/site) was carried out. The results show that both treatments blocked the effect of ConBr. Furthermore, the coadministration of subeffective doses of the NMDA receptor antagonist MK-801 (0.001mg/kg, i.p.) or ketamine (0.1mg/kg, i.p.; NMDA receptor antagonist) and ConBr (0.1μg/site, i.c.v.) caused a synergistic reduction in immobility time. In order to verify the dependence of the l-arginine–NO–cGMP pathway, on the effect of ConBr in the FST, a pretreatment with the NO precursor, l-arginine (750mg/kg, i.p.), or the PDE5 inhibitor, sildenafil (5mg/kg, i.p.), was performed. Both drugs abolished the antidepressant-like action of ConBr. Finally, the administration of subeffective doses of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 30pmol/site, i.c.v.) and ConBr (0.1μg/site, i.c.v.) produced a synergistic antidepressant-like effect in the FST. Taken together, the results suggest that the antidepressant-like effect of ConBr in the FST involves NMDA receptor inhibition and reduction in NO and cGMP synthesis. [Copyright &y& Elsevier]

Highlights: [•] A new lectin from Bauhinia ungulata seeds (BUL) was purified and characterized. [•] BUL showed antifungal activity against phytopathogenic species. [•] BUL exhibits antiproliferative effect against cells of human colon adenocarcinoma. [ABSTRACT FROM AUTHOR]

Highlights: [•] ConA, ConBol and ConBr bound to bovine sperm in different manners. [•] These lectins did not affect sperm viability or lipid peroxidation. [•] The three lectins induced a decrease in sperm motility and an increase in ROS production. [•] The three lectins reduced the embryo cleavage ratio but not the blastocyst formation ratio. [Copyright &y& Elsevier]

Abstract: The excitotoxicity induced by excessive activation of the glutamatergic neurotransmission pathway is involved in several neuropathologies. In this sense, molecules that prevent the release of glutamate or the excessive activation of its receptors can be useful in preventing the neuronal cell death observed in these diseases. Lectins are proteins capable of reversible binding to the carbohydrates in glycoconjugates, and some have been used in the study and purification of glutamate receptors. ConBr is a mannose/glucose-binding lectin purified from Canavalia brasiliensis seeds. In the present study, we aimed to evaluate the neuroprotective activity of ConBr against glutamate-induced excitotoxicity. Hippocampal slices were isolated from adult male mice and incubated for 6h in Krebs saline/DMEM buffer alone (control), in the presence of glutamate or glutamate plus ConBr. The phosphorylation of Akt and mitogen activated protein kinases (MAPKs) such as ERK1/2, p38MAPK and JNK1/2/3 was evaluated with western blotting. The results indicate that glutamate provoked a reduction in the hippocampal slice viability (−25%), diminished the phosphorylation of Akt and augmented p38MAPK and ERK1 phosphorylation. No changes were observed in the phosphorylation of JNK1/2/3 or ERK2. Notably, ConBr, through a mechanism dependent on carbohydrate interaction, prevented the reduction of cell viability and Akt phosphorylation induced by glutamate. Furthermore, in the presence of the PI3K inhibitor LY294002, ConBr was unable to reverse glutamate neurotoxicity. Taken together, our data suggest that the neuroprotective effect of ConBr against glutamate neurotoxicity requires oligosaccharide interaction and is dependent on the PI3K/Akt pathway. [Copyright &y& Elsevier]

Abstract: Vatairea guianensis seeds, a typical plant from the Brazilian Amazon region that belongs to the Dalbergieae tribe, possess a lectin that was isolated by precipitation with solid ammonium sulfate followed by guar gum affinity chromatography. This lectin was named VGL. The V. guianensis lectin strongly agglutinated rabbit erythrocytes and was inhibited by d-galactose and d-galactose-derived sugars, especially N-acetyl-d-galactosamine. VGL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with ethylenediamine tetraacetic acid (EDTA). In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the purified VGL exhibited an electrophoretic profile consisting of a major 30–32kDa double band, which is termed the alpha-chain, and two minor components of 18 and 15kDa, which are referred to as the beta- and gamma-chains, respectively. An analysis using electrospray ionization mass spectrometry also indicated that purified VGL contains a mixture of chains with molecular weights of 28,437±2, 14,952±2 and 12,332±2. The complete amino acid sequence of VGL, as determined using tandem mass spectrometry, consists of 239 amino acid residues. VGL is a glycoprotein exhibiting high similarity in primary structure to other lectins from evolutionarily related plants, such as Vatairea macrocarpa lectin and lectins belonging to the Sophoreae tribe. VGL exhibits vasorelaxant activity in contracted rat aortas, an effect that is strictly dependent on the endothelium and involves nitric oxide and the lectin domain. [Copyright &y& Elsevier]

Abstract: The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological properties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site–ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration. [Copyright &y& Elsevier]

Abstract: Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position. [Copyright &y& Elsevier]

Abstract: Hypnea cervicornis agglutinin (HCA), a lectin isolated from the red marine alga has been previously shown to have an antinociceptive effect. In the present study in rats, mechanisms of action of HCA were addressed regarding mechanical hypernociception induced by carrageenan, ovalbumin (as antigen), and also by prostaglandin E2 in rats. The lectin administered intravenously inhibited carrageenan- and antigen-induced hypernociception at 1, 3, 5 and 7h. This inhibitory effect was completely prevented when lectin was combined with mucin, demonstrating the role of carbohydrate-binding sites. The inhibition of inflammatory hypernociception by HCA was associated with the prevention of neutrophil recruitment to the plantar tissue of rats but was not associated with the inhibition of the release of pro-hypernociceptive cytokines (TNF-α, IL-1β and CINC-1). HCA also blocked mechanical hypernociception induced by PGE2, which was prevented by the administration of nitric oxide synthase inhibitors. These results were corroborated by the increased circulating levels of NO metabolites following HCA treatment. These findings suggest that the anti-hypernociceptive effects of HCA are not associated with the inhibition of pro-inflammatory cytokine production. However, these effects seem to involve the inhibition of neutrophil migration and also the increase in NO production. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125–1000μg/cavity), and at 2–96h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500μg/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-α, IL-1β and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31μg/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1β, TNF-α and CINC-1. [Copyright &y& Elsevier]

Abstract: Knowledge of the different patterns of gene expression along the male reproductive tract can assist in understanding the physiological processes of species-specific reproduction in mammals. In the present work, expression profiles of buck spermadhesin (bodhesin) genes along the reproductive tract by qRT-PCR were investigated. Total RNA from the seminal vesicle, testis, epididymis, bulbourethral gland and ductus deferens were reverse transcribed and the cDNA produced was submitted to qRT-PCR. For each homologous bodhesin gene, namely Bdh-1, Bdh-2 and Bdh-3, sets of specific primers and recombinant plasmids were prepared for gene quantification. In buck seminal vesicles, Bdh-2 is the homologue predominantly expressed, with a copy number on the order of millions of times more than Bdh-1 and thousand times more than Bdh-3. The copy number of Bdh-3 mRNA is only 10-fold greater than that of Bdh-1. Bodhesin transcripts were detected in all tissues examined, except in ductus deferens. The quantitative analysis also demonstrated clearly the differential gene expression of spermadhesin in bulbourethral gland. The striking differences in bodhesin gene expression indicate that each isoform could have a specific biological function in the buck genital tract, which deserves further detailed studies. [Copyright &y& Elsevier]

Abstract: This study investigates the action of the central administration of the lectins isolated from Canavalia brasiliensis seeds (ConBr) and from Canavalia ensiformes seeds, (Concanavalin A, ConA) in the forced swimming test (FST) in mice. ConBr (1–10 μg/site, i.c.v.), but not ConA, produced a decrease in the immobility time in the FST (observed at the time points 15, 30, 60 and 120 min after the injection), without changing the locomotor activity in the open‐field test. The effect of ConBr in the FST was dependent on its protein structure integrity. ConBr (0.1 μg/site, i.c.v.) caused a potentiation of the action of fluoxetine, a selective 5‐HT reuptake inhibitor. The anti‐immobility effect elicited by ConBr (10 μg/site, i.c.v.) in the FST was prevented by the pretreatment of mice with pindolol (32 mg/kg, a 5‐HT1A/1B receptor/β‐adrenoceptor antagonist), NAN‐190 (0.5 mg/kg, a 5‐HT1A receptor antagonist), ketanserin (5 mg/kg, a 5‐HT2A/2C receptor antagonist), sulpiride (50 mg/kg, a D2 receptor antagonist) or yohimbine (1 mg/kg, an α2‐adrenoceptor antagonist), but not with SCH 23390 (0.05 mg/kg, a D1 receptor antagonist) or prazosin (1 mg/kg, an α1‐adrenoceptor antagonist). These results indicate that the antidepressant‐like effect of ConBr in the FST is dependent on its interaction with the serotoninergic (via 5‐HT1A and 5‐HT2), noradrenergic (via α2‐adrenoceptors) and dopaminergic (via D2 receptors) systems. Considering the presence of lectins in the brain and based on the results, it will be important to determine a possible role of endogenous lectins in the modulation of the central nervous system function. [Copyright &y& Elsevier]

Abstract: In the present study we evaluated Canavalia brasiliensis (ConBr), Pisum arvense (PAA) and Artocarpus integrifolia (KM+) lectins as immunostimulatory molecules in vaccination against Leishmania amazonensis infection. Although they induced IFN-γ production, the combination of the lectins with SLA antigen did not lead to lesion reduction. However, parasite load was largely reduced in mice immunized with KM+ lectin and SLA. KM+ induced a smaller inflammatory reaction in the air pouch model and was able to inhibit differentiation of dendritic cells (BMDC), but to induce maturation by enhancing the expression of MHC II, CD80 and CD86. These observations indicate the modulatory role of plant lectins in leishmaniasis vaccination may be related to their action on the initial innate response. [Copyright &y& Elsevier]

Abstract: Arum maculatum agglutinin (AMA) is a monocot lectin isolated from tubers of Arum maculatum L. (Araceae) which exhibits different specificity towards oligo-mannosidic-type and N-acetyllactosaminic-type glycans. We have investigated the effect of this lectin on the cells of the immune system. Models of neutrophil migration in vivo, neutrophil chemotaxis in vitro and macrophage cultures were used to study the lectin inflammatory activity. When administered into rat peritoneal cavities, AMA (80, 200 and 500μg/mL/cavity) induced significant and dose-dependent neutrophil migration. This effect was inhibited by incubation with α-methyl-d-mannoside. A 83% depletion in the number of resident cells following peritoneal lavage did not reduce the AMA-induced neutrophil migration, as compared to sham animals (not washed). However, pre-treatment with 3% thioglycolate which increases the peritoneal macrophage population by 236%, enhanced the neutrophil migration induced by AMA (200μg/mL/cavity) (119%, p &#60;0.05). Reduction of peritoneal mast cell population by chronic treatment of cavities with compound 48/80 did not modify AMA-induced neutrophil migration. The neutrophil chemotaxy assay in vitro shows that the lectin (300μg/mL) induces neutrophil chemotaxy (368% p &#60;0.05) compared to RPMI. Finally, injection into peritoneal cavities of supernatants from macrophage cultures obtained after stimulation with AMA (300μg/mL) enhanced neutrophil migration (110% p &#60;0.05). Summarizing, our data suggest that A. maculatum agglutinin presents pro-inflammatory activity, inducing neutrophil migration by two ways, one which is independent on resident cells and another one dependent on the presence of these cells. [Copyright &y& Elsevier]

To explore the pathways by which lectins induce an inflammatory response, the lectin from Vatairea macrocarpa (VML) seeds was used to induce neutrophil migration in rats. The lectin was shown to cause cell migration, with the effect partially blocked when galactose was added to inhibit lectin activity. Neutrophil migration was also reduced when peritoneal cavity of the animals was depleted of their resident cells beforehand, suggesting that neutrophil migration was mediated by an indirect mechanism. Pre-treatment of rats with thioglycollate increased recruitment of neutrophils while depletion of mast cells by the addition of compound 48/80 had little effect on neutrophil infiltration, suggesting the involvement of macrophages in the inflammatory process induced by the lectin. Inhibition of the cyclooxigenase, leukotriene and PAF activities by indomethacin, MK886 and BN50730, respectively, did not modify the pro-inflammatory effect previously observed. However, dexamethasone and thalidomide significantly reduced the population of neutrophils in the peritoneal cavity after lectin injection. The present study suggests that the effects produced by a galactose-binding lectin do not involve lipoxygenase, cyclooxygenase or PAF mediators that are well known to be involved in the inflammatory process. The blocking actions of dexamethasone and thalidimide suggest that as yet unidentified pro-inflammatory mediators are involved. [Copyright &y& Elsevier]