ConBr, A Lectin Purified from the Seeds of Canavalia brasiliensis, Protects Against Ischemia in Organotypic Culture of Rat Hippocampus: Potential Implication of Voltage-Gated Calcium Channels.

Lectins are proteins that bind cellular glycans and can modulate various neuronal functions. We have evaluated the neuroprotective effect of ConBr, a lectin purified from the seeds of Canavalia brasiliensis in a model of rat organotypic hippocampal cultures (OHCs) exposed to oxygen and glucose deprivation (OGD). OGD for 15 min followed by 24 h re-oxygenation significantly increased cell death, caused mitochondrial depolarization and increased reactive oxygen species (ROS) in CA1 region of OHCs. ConBr (0.1 µg/mL) added during the re-oxygenation period counteracted cell death, mitochondrial depolarization and overproduction of ROS induced by OGD. Moreover, ConBr restored the levels of Akt and ERK1 phosphorylation that were reduced by OGD. Modulation of intracellular Ca2+ by ConBr was evaluated in isolated hippocampal neurons loaded with the fluorescent calcium dye Fluo-4/AM. ConBr (0.1 and 1 µg/mL) reduced by 25-30 % the Ca2+ increment induced by 70 mM K+. A sub effective concentration of ConBr (0.01 µg/mL) together with a sub effective concentration of the L-type calcium channel antagonist nifedipine (0.3 µM) conferred a synergic neuroprotective effect in OHCs subjected to OGD. In conclusion, ConBr provides OHCs neuroprotection against OGD. The mechanism was not fully addressed but it may involve modulation of L-type voltage-gated Ca2+ channels by ConBr.

Inhibition of initial adhesion of oral bacteria through a lectin from Bauhinia variegata L. var. variegata expressed in Escherichia coli.

AIMS: The aim of the present work was to study the in vitro effect of native and recombinant Bauhinia variegata var. variegata lectins in inhibiting early adhesion of Streptococcus mutans, Streptococcus sanguis and Streptococcus sobrinus to experimentally acquired pellicle. METHODS AND RESULTS: Native lectin from B. variegata (BVL) was purified by affinity chromatography of extract of seeds. The recombinant lectin (rBVL-I) was expressed in E. coli strain BL21 (DE3) from a genomic clone encoding the mature B. variegata lectin gene using the vector pAE-bvlI. Recombinant protein deposited in inclusion bodies was solubilized and subsequently purified by affinity chromatography. The rBVL-I was compared to BVL for agglutination of erythrocytes and initial adherence of oral bacteria on a saliva-coated surface. The results revealed that rBVL-I acts similarly to BVL for agglutination of erythrocytes. Both lectins showed adhesion inhibition effect on Step. sanguis, Step. mutans and Step. sobrinus. CONCLUSION: We report, for the first time, the inhibition of early adhesion of oral bacteria by a recombinant lectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the proposed biotechnological application of lectins in a strategy to reduce development of dental caries by inhibiting the initial adhesion and biofilm formation.

Cloning and molecular modeling of Litopenaeus vannamei (Penaeidae) C-type lectin homologs with mutated mannose binding domain-2.

C-type lectins are animal proteins that contain at least one carbohydrate recognition domain (CRD) capable of mediating sugar and calcium binding. Carbohydrate recognition is directly required for some biological functions, including the innate immune response. We cloned two novel C-type lectin (CTL) precursors from the commercial marine shrimp Litopenaeus vannamei. The cloned cDNAs encompass ORFs of 1044 nucleotides and encode highly similar two-domain polypeptides of 347 residues. The predicted proteins, LvCTL-br1 and -br2, contain the consensus triad that recognizes galactose (-GlnProAsp-) in CRD1 but also contain a mutated mannose-binding site (-GluProAsn-) in the second domain (CRD2). Phylogenetic analysis of LvCTL-br1 and -br2 and hundreds of CTL-like domain-containing proteins have allowed grouping of penaeid shrimp CTLs into three functional clusters. Reverse transcription coupled to PCR indicated that LvCTL-br1 expression is induced in shrimp gills upon IHHNV infection. Computational molecular modeling of LvCTL-br1 and -br2 revealed that three amino acid substitutions in CRD1 occur near the sugar binding site. Also, the 3-D models show a long loop of LvCTL-br1 CRD2 that might accommodate complex sugars. The structural data, evolutionary history and functional analysis support the hypothesis that gene duplication and accelerated evolution have caused functional diversification of penaeid shrimp C-type lectins.

Cloning and molecular modeling of Litopenaeus vannamei (Penaeidae) C-type lectin homologs with mutated mannose binding domain-2

C-type lectins are animal proteins that contain at least one carbohydrate recognition domain (CRD) capable of mediating sugar and calcium binding. Carbohydrate recognition is directly required for some biological functions, including the innate immune response. We cloned two novel C-type lectin (CTL) precursors from the commercial marine shrimp Litopenaeus vannamei. The cloned cDNAs encompass ORFs of 1044 nucleotides and encode highly similar two- domain polypeptides of 347 residues. The predicted proteins, LvCTL-br1 and -br2, contain the consensus triad that recognizes galactose (-GlnProAsp-) in CRD1 but also contain a mutated mannose-binding site (-GluProAsn-) in the second domain (CRD2). Phylogenetic analysis of LvCTL-br1 and -br2 and hundreds of CTL-like domain-containing proteins have allowed grouping of penaeid shrimp CTLs into three functional clusters. Reverse transcription coupled to PCR indicated that LvCTL-br1 expression is induced in shrimp gills upon IHHNV infection. Computational molecular modeling of LvCTL-br1 and -br2 revealed that three amino acid substitutions in CRD1 occur near the sugar binding site. Also, the 3-D models show a long loop of LvCTL-br1 CRD2 that might accommodate complex sugars. The structural data, evolutionary history and functional analysis support the hypothesis that gene duplication and accelerated evolution have caused functional diversification of penaeid shrimp C-type lectins.

Crystal structure of Bn IV in complex with myristic acid: a Lys49 myotoxic phospholipase A2 from Bothrops neuwiedi venom.

The LYS49-PLA2s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA2 is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111-121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA2 was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH3(CH2)12COOH) and its overall structure was refined at 2.2 Å resolution. The Bn IV crystals belong to monoclinic space group P21 and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 Å. The biological assembly is a "conventional dimer" and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes.

Effects of low molecular weight sulfated galactan fragments from Botryocladia occidentalis on the pharmacological and enzymatic activity of sPLA2 from Crotalus durissus cascavella.

Low molecular weight fragments of sulfated galactans (Boc-5 and Boc-10) from the red algae Botryocladia occidentalis significantly inhibited Crotalus durissus cascavella sPLA2 enzymatic activity. Equimolar ratios of sPLA2 to Boc-5 or Boc-10 resulted in allosteric inhibition of sPLA2. Under the conditions tested, we observed that both Boc-5 and Boc-10 strongly decreased edema, myonecrosis, and neurotoxicity induced by native sPLA2.

Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA.

Low purification efficiency and incomplete characterization of male goat (buck) spermadhesins (Bdhs) prompted us to develop an effective system to produce recombinant Bdhs (rBdhs). Bdh-2 cDNA was inserted into a prokaryotic expression plasmid, pTrcHis TOPO. The pTrcHis-Bdh-2 system was constructed to produce a His(6) fusion protein in Escherichia coli Top10 cells. Recombinant clones were selected by growth in ampicillin-enriched medium, PCR amplification and nucleotide sequencing. The inserted cDNA was completely identified and recombinant protein synthesis was monitored by SDS-PAGE, followed by immunoblotting with monoclonal anti-His antibody. Expression of insoluble rBdh-2 was achieved at 0.1 to 2.0 mM IPTG, after 2 to 6 h of induction. Significantly increased production of rBdh-2 (P < 0.01) occurred with 1.5 mM IPTG after 2 h of induction, and with 0.3 mM IPTG after 4 h in culture. Among the induction times investigated, a period of 6 h gave the lowest levels of rBdh-2 production; with a 6-h incubation, there were no significant differences in rBdh-2 production for the various concentrations of IPTG tested (P > 0.05). The apparent molecular weight of rBdh-2 was 15.85 +/- 0.09 kDa, calculated by image analysis of membranes. This is similar to the theoretical molecular weight of 15.5 kDa predicted from the nucleotide sequence. Prior to this study, expression of recombinant goat spermadhesin had never been reported. Thus, an effective prokaryotic rBdh-2 expression system was developed in order to provide an adequate tool for studying biofunctions of goat spermadhesins.

Adsorption of ascorbic acid on the C60 fullerene.

Adsorption of ascorbic acid (AsA) on C60 is investigated using classical molecular mechanics and density functional theory (DFT). Classical annealing was performed to explore the space of molecular configurations of ascorbic acid adsorbed on C60, searching for optimal geometries. From the structure with the smallest total energy, 10 initial configurations were prepared by applying rotations of 90 degrees about three orthogonal axes. Each one of these configurations was optimized using DFT (for both LDA and GGA exchange-correlation functionals), and an estimate of their total and adsorption energies was found. Different configurations have minimal adsorption energies (defined here as the total energy of the adsorbate minus the total energy of the separate molecules) from -0.54 to -0.10 eV, with distinct optimal distances between the AsA and C60 centers of mass. According to a Hirshfeld population analysis, AsA is, in general, an acceptor of electrons from C60. Our results demonstrate the feasibility of noncovalent functionalization of C60 with AsA and provide minimal energy values for the several different configurations investigated. These results should be considered in reactions as a possible way to prevent against the oxidative damage and toxicity of C60. The beneficial effects of using AsA-C60 includes its action when administered together with levodopa, against the neurotoxicity generated by levodopa isolated, which opens new strategies for the Parkinson's disease treatment.

Crystal structure of Dioclea rostrata lectin: insights into understanding the pH-dependent dimer-tetramer equilibrium and the structural basis for carbohydrate recognition in Diocleinae lectins.

The legume lectins from the subtribe Diocleinae, often referred to as concanavalin A-like lectins, are a typical example of highly similar proteins that show distinct biological activities. The pH-dependent oligomerization that some of these lectins undergo and the relative position of amino acids within the carbohydrate-binding site are factors that have been reported to contribute to these differences in the activities of Diocleinae lectins. In the present work, we determined the amino acid sequence and the crystal structure of the lectin of Dioclea rostrata seeds (DRL), with the aim of investigating the structural bases of the different behavior displayed by this lectin in comparison to other Diocleinae lectins and determining the reason for the distinct pH-dependent dimer-tetramer equilibrium. In addition, we discovered a novel multimeric arrangement for this lectin.

Purification of a PHA-like chitin-binding protein from Acacia farnesiana seeds: a time-dependent oligomerization protein.

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 +/- 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

In vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins.

AIMS: The initial colonization of the tooth by streptococci involves their attachment to adsorbed components of the acquired pellicle. Avoiding this adhesion may be successful in preventing caries at early stages. Salivary mucins are glycoproteins that when absorbed onto hydroxyapatite may provide binding sites for certain bacteria. Algal lectins may be especially interesting for oral antiadhesion trials because of their great stability and high specificity for mucins. This work aimed to evaluate the potential of two algal lectins to inhibit the adherence of five streptococci species to the acquired pellicle in vitro. METHODS AND RESULTS: The lectins used were extracted from Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL). Fluorescence microscopy was applied to visualize the ability of fluorescein isothiocyanate-labelled lectins to attach to the pellicle and revealed a similar capability for both lectins. Streptococcal adherence assays were performed using saliva-coated microtitre plates. BSL inhibited more than 75% of Streptococcus sanguis, Streptococcus mitis, Streptococcus sobrinus and Streptococcus mutans adherence, achieving 92% to the latter. BTL only obtained statistically significant results on S. mitis and S. sobrinus, whose adherence was decreased by 32.5% and 54.4%, respectively. CONCLUSION: Algal lectins are able to inhibit streptococcal adherence. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the proposed application of lectins in antiadhesion therapeutics.

Identification of lamivudine conformers by Raman scattering measurements and quantum chemical calculations.

Characterization of nucleoside and non-nucleoside human immunodeficiency virus (HIV) reverse transcriptase inhibitors conformers, NRTIs and NNRTIs, respectively, is fundamental for an improved treatment of infected individuals. Three conformers in lamivudine I powder are quickly identified in this work by assignment of some Raman peaks to their vibrational frequencies, as obtained by first principles quantum chemical calculations. The method is proposed as a practical procedure for non-destructive identification, analysis, and process monitoring of NRTIs and NNRTIs conformers.

In vitro inhibition of Streptococci binding to enamel acquired pellicle by plant lectins.

AIM: Initial colonization of the tooth surface by streptococci involves the attachment of these bacteria to adsorbed salivary components of the acquired pellicle. In dental biofilm this adhesion may also involve lectin-like components, present on the surface of the organisms, which bind to complementary carbohydrates on the surface of the tooth. Therefore, this work aimed to evaluate the potential of six lectins, extracted from seeds of Leguminosae family members, to inhibit the adherence of five streptococci species to acquired pellicle in vitro. METHODS AND RESULTS: The lectins used in this work were extracted from Canavalia ensiformis, Canavalia brasiliensis, Dioclea violacea, Dioclea grandiflora, Cratylia floribunda and Vatairea macrocarpa. Fluorescence micrography was employed to visualize the ability of FITC-labeled lectins to attach to acquire pellicle. Adherence inhibition was performed on saliva-coated microtiter plates at which lectins solutions were previously incubated followed by incubation with the oral streptococci. Glucose-mannose specific lectins attached to acquired pellicle with high intensity, while galactose specific lectins, from V. macrocarpa, exhibits low intensity attachment. CONCLUSIONS: All lectins were able to inhibit the adherence of the microorganisms tested (p < 0.01). SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that lectins may be useful in anti adhesion therapeutics.

Ion-exchange chromatography used to isolate a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma

Mammalian seminal plasma contains among others, proteins called spermadhesins, which are the major proteins of boar and stallion seminal plasma. These proteins appear to be involved in capacitation and sperm-egg interaction. Previously, we reported the presence of a protein related to spermadhesins in goat seminal plasma. In the present study, we have further characterized this protein, and we propose ion-exchange chromatography to isolate this seminal protein. Semen was obtained from four adult Saanen bucks. Seminal plasma was pooled, dialyzed against distilled water and freeze-dried. Lyophilized proteins were loaded onto an ion-exchange chromatography column. Dialyzed-lyophilized proteins from the main peak of DEAE-Sephacel were applied to a C2/C18 column coupled to an RP-HPLC system, and the eluted proteins were lyophilized for electrophoresis. The N-terminal was sequenced and amino acid sequence similarity was determined using CLUSTAL W. Additionally, proteins from DEAE-Sephacel chromatography step were dialyzed and submitted to a heparin-Sepharose high-performance liquid chromatography. Goat seminal plasma after ion-exchange chromatography yielded 6.47 +/- 0.63 mg (mean +/- SEM) of the major retained fraction. The protein was designated BSFP (buck seminal fluid protein). BSFP exhibited N-terminal sequence homology to boar, stallion and bull spermadhesins. BSFP showed no heparin-binding capabilities. These results together with our previous data indicate that goat seminal plasma contains a protein that is structurally related to proteins of the spermadhesin family. Finally, this protein can be efficiently isolated by ion-exchange and reverse-phase chromatography.

Isolation and characterization of a new agglutinin from the red marine alga Hypnea cervicornis J. Agardh.

The biochemical characterization of a new lectin (Hypnea cervicornis agglutinin or HCA) isolated from the Brazilian red alga H. cervicornis is reported. The haemagglutinating activity of the lectin was only inhibited by the glycoprotein porcine stomach mucin at a minimum inhibitory concentration of 19 microg x mL(-1). No haemagglutination inhibition was detected after the addition of simple sugars. The MALDI-TOF molecular masses of native and reduced and carbamidomethylated HCA were, respectively, 9196.6 Da and 9988.2 Da, indicating that the primary structure of the protein is crosslinked by 7 disulfide bonds. This unusual structural feature among lectins, along with its N-terminal sequence and amino-acid composition, clearly shows that HCA belongs to a protein family distinct from the isolectins Hypnin A1 and A2 isolated from the related Japanese alga Hypnea japonica. On the other hand, HCA displayed a high degree of similarity to the agglutinin from the Brazilian species Hypnea musciformis. Our data indicate the occurrence of structural diversity among lectins of closely related species living in distant ecosystems, i.e., the Pacific coast of Japan and the Atlantic coast of Brazil, and support the hypothesis that the lectin content (lectinome) might serve as a biomarker for taxonomical purposes.

Crotacetin, a novel snake venom C-type lectin, is homolog of convulxin

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins, which are able to interfere with hemostasis. They share significant similarity in their primary structures with C-type lectins of other animals, and also present a conserved carbohydrate recognition domain (CRD). A very well studied sv C-type lectin is the heterodimeric toxin, convulxin (CVX), from the venoms of South American rattlesnakes, Crotalus durissus terrificus and C. d. cascavella. It consists of two subunits, alfa (CVXa, 13.9 kDa) and beta (CVXb, 12.6 kDa), joined by inter and intra-chain disulfide bounds, and is arranged in a tetrameric a4b4 conformation. Convulxin is able to activate platelet and induce their aggregation by acting via p62/GPVI collagen receptor. Several cDNA precursors, homolog of CVX subunits, were cloned by PCR homology screening. As determined by computational analysis, one of them, named crotacetin b subunit, was predicted as a polypeptide with a tridimensional conformation very similar to other subunits of convulxin-like snake toxins. Crotacetin was purified from C. durissus venoms by gel permeation and reverse phase high performance liquid chromatography. The heterodimeric crotacetin is expressed in the venoms of several C. durissus subspecies, but it is prevalent in the venom of C. durissus cascavella. As inferred from homology modeling, crotacetin induces platelet aggregation but noticeably exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria

Anti-inflammatory and antimicrobial effect of lectin from Lonchocarpus sericeus seeds in an experimental rat model of infectious peritonitis.

We have investigated the anti-inflammatory and antimicrobial effect of the lectin from Lonchocarpus sericeus seeds (LSL) in a model of infectious peritonitis in adult Wistar rats. Animals were treated with saline or LSL (10 mg kg(-1), i.v) immediately and 6 h after the induction of peritonitis via cecal ligation and single puncture. Twelve hours after surgery, animals were killed and the infectious process was monitored by total and differential count of cells from blood and peritoneal washing liquid, adenosine deaminase activity, antibiogram and the number of viable bacteria of the peritoneal cavity. LSL treatment decreased the inflammatory response evoked by the induction of peritonitis, as seen by the inhibition of neutrophil migration into peritoneal cavities, leucocytosis and reduction of adenosine deaminase activity in the peritoneal fluid. All these effects were reversed by the lectin association to N-acetyl-glucosamine. LSL in-vitro did not show any antimicrobial action, but promoted a marked decrease of the viable bacterial population in peritoneal cavities. In conclusion, LSL inhibited the inflammatory response and the bacterial colonization of infectious peritonitis in rats.

Differential effect of plant lectins on mast cells of different origins.

Histamine release induced by plant lectins was studied with emphasis on the carbohydrate specificity, external calcium requirement, metal binding sites, and mast cell heterogeneity and on the importance of antibodies bound to the mast cell membrane to the lectin effect. Peritoneal mast cells were obtained by direct lavage of the rat peritoneal cavity and guinea pig intestine and hamster cheek pouch mast cells were obtained by dispersion with collagenase type IA. Histamine release was induced with concanavalin A (Con A), lectins from Canavalia brasiliensis, mannose-specific Cymbosema roseum, Maackia amurensis, Parkia platycephala, Triticum vulgaris (WGA), and demetallized Con A and C. brasiliensis, using 1-300 microg/ml lectin concentrations applied to Wistar rat peritoneal mast cells, peaking on 26.9, 21.0, 29.1, 24.9, 17.2, 10.7, 19.9, and 41.5%, respectively. This effect was inhibited in the absence of extracellular calcium. The lectins were also active on hamster cheek pouch mast cells (except demetallized Con A) and on Rowett nude rat (animal free of immunoglobulins) peritoneal mast cells (except for mannose-specific C. roseum, P. platycephala and WGA). No effect was observed in guinea pig intestine mast cells. Glucose-saturated Con A and C. brasiliensis also released histamine from Wistar rat peritoneal mast cells. These results suggest that histamine release induced by lectins is influenced by the heterogeneity of mast cells and depends on extracellular calcium. The results also suggest that this histamine release might occur by alternative mechanisms, because the usual mechanism of lectins is related to their binding properties to metals from which depend the binding to sugars, which would be their sites to bind to immunoglobulins. In the present study, we show that the histamine release by lectins was also induced by demetallized lectins and by sugar-saturated lectins (which would avoid their binding to other sugars). Additionally, the lectins also released histamine from Rowett nude mast cells that are free of immunoglobulins.

Differential effect of plant lectins on mast cells of different origins

Histamine release induced by plant lectins was studied with emphasis on the carbohydrate specificity, external calcium requirement, metal binding sites, and mast cell heterogeneity and on the importance of antibodies bound to the mast cell membrane to the lectin effect. Peritoneal mast cells were obtained by direct lavage of the rat peritoneal cavity and guinea pig intestine and hamster cheek pouch mast cells were obtained by dispersion with collagenase type IA. Histamine release was induced with concanavalin A (Con A), lectins from Canavalia brasiliensis, mannose-specific Cymbosema roseum, Maackia amurensis, Parkia platycephala, Triticum vulgaris (WGA), and demetallized Con A and C. brasiliensis, using 1-300 æg/ml lectin concentrations applied to Wistar rat peritoneal mast cells, peaking on 26.9, 21.0, 29.1, 24.9, 17.2, 10.7, 19.9, and 41.5 percent, respectively. This effect was inhibited in the absence of extracellular calcium. The lectins were also active on hamster cheek pouch mast cells (except demetallized Con A) and on Rowett nude rat (animal free of immunoglobulins) peritoneal mast cells (except for mannose-specific C. roseum, P. platycephala and WGA). No effect was observed in guinea pig intestine mast cells. Glucose-saturated Con A and C. brasiliensis also released histamine from Wistar rat peritoneal mast cells. These results suggest that histamine release induced by lectins is influenced by the heterogeneity of mast cells and depends on extracellular calcium. The results also suggest that this histamine release might occur by alternative mechanisms, because the usual mechanism of lectins is related to their binding properties to metals from which depend the binding to sugars, which would be their sites to bind to immunoglobulins. In the present study, we show that the histamine release by lectins was also induced by demetallized lectins and by sugar-saturated lectins (which would avoid their binding to other sugars). Additionally, the lectins also released histamine from Rowett nude mast cells that are free of immunoglobulins.

Lectin of Pisum arvense seeds induces in-vivo and in-vitro neutrophil migration.

PAL is a glucose/mannose-specific lectin isolated from Pisum arvense seeds. Previously, we demonstrated the capacity of other leguminous lectins to induce oedema formation and neutrophil stimulation. To investigate the potential pro-inflammatory activity of PAL, we have studied its ability to induce neutrophil migration into peritoneal cavities of rats and neutrophil chemotaxis in-vitro. The role of resident cells and sugar residues on PAL activity was analysed. PAL or saline (control) were administered intraperitoneally to rats, and total and differential leucocyte (macrophages, neutrophils and mast cells) counts were performed. The role of resident cells on the PAL effect was evaluated using three strategies: reducing the total resident cell population by lavage of rat cavities with saline; increasing macrophage population by treating animals with thioglycolate; and depleting mast cell population by subchronic treatment of rats with compound 48/80. PAL induced in-vitro and in-vivo neutrophil migration. In-vivo, PAL (50, 100, 200 and 300 microg) significantly (P < 0.05) and dose-dependently increased neutrophil migration by 600, 740, 900 and 940%, respectively, showing maximal effect 4 h after injection. PAL induced mononuclear cell migration. The neutrophil stimulatory effect of PAL was potentiated in animals treated with both thioglycolate and compound 48/ 80. The indirect lectin chemotactic effect was shown in rats injected with supernatant from cultured macrophages stimulated by PAL. In conclusion, PAL was shown to exhibit in-vivo and in-vitro proinflammatory activity. The in-vivo effect seemed to occur by a dual mechanism that was independent, but also dependent, on resident cells.