Conformation of the primary binding loop folded through an intramolecular interaction contributes to the strong chymotrypsin inhibitory activity of the chymotrypsin inhibitor from Erythrina variegata seeds.
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We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina variegata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanaga et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not these four amino acid residues predominantly contribute to the strong inhibitory activity of ECI, they were simultaneously replaced by Ala. The results showed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable inhibitory activity (Ki, 5.6 x 10(-7) M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of the primary binding loop in ECI is essential for the inhibitory activity toward chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with lower chymotrypsin inhibitory activity, were constructed to determine whether the backbone structure of the primary binding loop of ECI was formed by the amino acid residues therein, or through an interaction between the primary binding loop and the residual structure designated as the "scaffold." A chimeric protein, ECI/ETIa, composed of the primary binding loop of ECI and the scaffold of ETIa showed weaker inhibitory activity (Ki, 1.3 x 10(-6) M) than ECI (Ki, 9.8 x 10(-8) M). In contrast, a chimera, ETIa/ECI, comprising the primary binding loop of ETIa and the scaffold of ECI inhibited chymotrypsin more strongly (Ki, 5.7 x 10(-7) M) than ETIa (Ki, 1.3 x 10(-6) M). These results indicate that the intramolecular interaction between the primary binding loop and the scaffold of ECI plays an important role in the strong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding loop of ECI contribute to both an increase in the association rate constant (kon) and a decrease in the dissociation rate constant (koff) for the ECI-chymotrypsin interaction, whereas the backbone structure of the primary binding loop mainly contributes to a decrease in the dissociation rate constant. (+info)
Structure/thermodynamics relationships of lectin-saccharide complexes: the Erythrina corallodendron case.
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Molecular dynamics (MD) simulations of Erythrina corallodendron lectin binding to a monosaccharide, alpha-galactose, and a disaccharide, N-acetyl lactosamine, have been performed in order to investigate the relationship between structure and thermodynamics. A simulated annealing protocol has been used to generate ensembles of structures for the two complexes, from which both qualitative and quantitative information on binding dynamics have been extracted. The ensembled averaged lectin-saccharide interaction enthalpy is equivalent for both sugars, whereas the calculation based on the X-ray structures does show a difference. Within large statistical errors, the calculated 'binding enthalpy' is also the same for the two systems. These errors arise largely from terms involving solvent and are a typical limitation of current MD simulations. Significant qualitative differences in binding between the two complexes are, however, observed over the ensembles. These could be important for unraveling the structure/thermodynamic relationship. Stated simply, there are a greater number of binding options available to the disaccharide compared to the monosaccharide. The implications of alternative binding states on thermodynamic parameters and the 'breaking of enthalpy-entropy compensation' are discussed. The role of solvent in lectin-saccharide complex formation is suggested to be significant. (+info)
Common architecture of the primary galactose binding sites of Erythrina corallodendron lectin and heat-labile enterotoxin from Escherichia coli in relation to the binding of branched neolactohexaosylceramide.
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The heat-labile enterotoxin from Escherichia coli (LT) is responsible for so-called traveller's diarrhea and is closely related to the cholera toxin (CT). Toxin binding to GM1 at the epithelial cell surface of the small intestine initiates the subsequent diarrheal disease. However, LT has a broader receptor specificity than CT in that it also binds to N-acetyllactosamine-terminated structures. The unrelated lectin from Erythrina corallodendron (ECorL) shares this latter binding property. The findings that both ECorL and porcine LT (pLT) bind to lactose as well as to neolactotetraosylceramide suggests a common structural theme in their respective primary binding sites. Superimposing the terminal galactose of the lactoses in the respective crystal structures of pLT and ECorL reveals striking structural similarities around the galactose despite the lack of sequence and folding homology, whereas the interactions of the penultimate GlcNAcb3 in the neolactotetraosylceramide differ. The binding of branched neolactohexaosylceramide to either protein reveals an enhanced affinity relative to neolactotetraosylceramide. The b3-linked branch is found to bind to the primary Gal binding pocket of both proteins, whereas the b6-linked branch outside this site provides additional interactions in accordance with the higher binding affinities found for this compound. While the remarkable architectural similarities of the primary galactose binding sites of pLT and ECorL point to a convergent evolution of these subsites, the distinguishing structural features determining the overall carbohydrate specificities are located in extended binding site regions. In pLT, Arg13 is thus found to play a crucial role in enhancing the affinity not only for N-acetyllactosamine-terminated structures but also for GM1 as compared to human LT (hLT) and CT. The physiological relevance of the binding of N-acetyllactosamine-containing glycoconjugates to LT and ECorL is briefly discussed. (+info)
Samsonia erythrinae gen. nov., sp. nov., isolated from bark necrotic lesions of Erythrina sp., and discrimination of plant-pathogenic Enterobacteriaceae by phenotypic features.
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Bacterial strains isolated from diseased erythrina (Erythrina sp.) trees in Martinique (French West Indies) were studied using phenotypic tests, 16S rDNA sequence analysis and DNA-DNA hybridization. Numerical analysis of phenotypic characteristics showed that these strains formed an homogeneous phenon among plant-pathogenic Enterobacteriaceae, and gave useful and updated information for the identification of these bacteria. Results of DNA-DNA hybridization indicated that strains from erythrina belonged to a discrete genomospecies (89-100% hybridization) and had low levels of DNA relatedness (2-33% hybridization) with reference strains of phytopathogenic Erwinia, Brenneria, Pectobacterium, Pantoea and Enterobacter species. 16S rDNA sequence analysis using three different methods revealed that the position of strain CFBP 5236T isolated from erythrina was variable in the different trees, so that strains from erythrina could not be assigned to any recognized genus. It is proposed that these strains are included in a new genus, Samsonia. The name Samsonia erythrinae is proposed for the new species. The G+C content of the DNA of the type strain, CFBP 5236T (= ICMP 13937T), is 57.0 mol%. (+info)
Lectin binding and endocytosis at the apical surface of human airway epithelia.
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The specificity of lectin binding to distinct saccharides makes them valuable reagents for investigation and identification of cells within complex tissues and potentially for delivery of agents into cells. Therefore we examined lectin binding to airway epithelia. We used an in vitro model of primary cultures of well-differentiated human airway epithelia and applied the lectins to the apical surface of living epithelia. This approach limited binding specifically to the extracellular surface of the apical membrane. Of 32 lectins studied, we found 15 that bound to the apical membrane. The pattern varied from diffuse binding to the surface of nearly all the cells, to binding to a small subset of the cells. Our data combined with earlier studies identify lectins that may be used to detect specific populations of epithelial cells. Because lectins may be used to deliver a variety of agents, including gene transfer vectors, to airway cells, we examined endocytosis of lectins. We found that several lectins bound to the apical surface were actively taken up into the cells. These data may be of value for studies of airway epithelial structure and may facilitate the targeting of the epithelial apical surface. (+info)
Effect of acute treatment with a water-alcohol extract of Erythrina mulungu on anxiety-related responses in rats.
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We investigated the effect of acute oral treatment with a water-alcohol extract of the inflorescence of Erythrina mulungu (EM, Leguminosae-Papilionaceae) (100, 200 and 400 mg/kg) on rats submitted to different anxiety models: the elevated T-maze (for inhibitory avoidance and escape measurements), the light/dark transition, and the cat odor test. These models were selected for their presumed capacity to demonstrate specific subtypes of anxiety disorders as recognized in clinical practice. Treatment with 200 mg/kg EM impaired avoidance latencies (avoidance 1 - 200 mg/kg EM: 18 +/- 7 s, control group: 40 +/- 9 s; avoidance 2 - 200 mg/kg EM: 15 +/- 4 s, control group: 110.33 +/- 38 s) in a way similar to the reference drug diazepam (avoidance 1: 3 +/- 0.79 s; avoidance 2: 3 +/- 0.76 s), without altering escape. Additionally, the same treatments increased the number of transitions (200 mg/kg EM: 6.33 +/- 0.90, diazepam: 10 +/- 1.54, control group: 2.78 +/- 0.60) between the two compartments and the time spent in the lighted compartment in the light/dark transition model (200 mg/kg EM: 39 +/- 7 s; diazepam: 61 +/- 9 s; control group: 14 +/- 4 s). The dose of 400 mg/kg EM also increased this last measurement (38 +/- 8 s). These results were not due to motor alterations since no significant effects were detected in the number of crossings or rearings in the arena. Furthermore, neither EM nor diazepam altered the behavioral responses of rats to a cloth impregnated with cat odor. These observations suggest that EM exerts anxiolytic-like effects on a specific subset of defensive behaviors, particularly those that have been shown to be sensitive to low doses of benzodiazepines. (+info)
Exploitation of Erythrina dominguezii Hassl. (Fabaceae) nectar by perching birds in a dry forest in western Brazil.
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Among the vertebrate pollinated plants, the genus Erythrina includes tree species in which birds are the pollen vectors. Two groups in this genus may be distinguished: a) the hummingbird, and b) the perching bird pollinated species. Erythrina dominguezii is included in the second group and occurs in deciduous/semi-deciduous forests in the southwestern neotropics. I studied the exploitation of Erythrina dominguezii nectar by perching birds in a dry forest in western Brazil. Six perching bird species from two distinct groups (Psittacidae: Brotogeris chiriri, Nandayus nenday, Aratinga acuticaudata; Icterinae: Psarocolius decumanus, Icterus cayanensis, I. icterus) consumed its nectar. The two most important consumers were Brotogeris chiriri (51.5% of the flowers visited by birds) and Psarocolius decumanus (20%). While B. chiriri was a flower predator, P. decumanus removed the nectar without damaging the flowers which it opened by inserting its large bill between the standard and the keel. Nandayus nenday, Aratinga acuticaudata, and I. icterus exploited the nectar like P. decumanus, and presumably also contributed to pollen transfer. As the flowering in E. dominguezii was intense and synchronous during the dryest period of the year, and its nectar was highly consumed by birds, the present data suggest that the nectar of this species may be important as an alternative resource to frugivorous/omnivorous birds when other resources are scarce. (+info)
Antinociceptive activities of the hydroalcoholic extracts from Erythrina velutina and Erythrina mulungu in mice.
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This work studied the antinociceptive effects of the hydroalcoholic extracts (HAEs) from Erythrina velutina (Ev) and Erythrina mulungu (Em) in three experimental models of nociception in mice. The extract was administered intraperitoneally to female mice at the doses of 200 and 400 mg/kg. Inhibitions of abdominal contractions were observed with the doses of 200 (88.6%; 86.8%) and 400 (95.5%; 83.5%) mg/kg of E. velutina and E. mulungu, respectively, as compared to controls. E. velutina and E. mulungu, at both doses, reduced the nociception produced by formalin in the 1st and 2nd phases and this effect was not reversed by the pretreatment with naloxone. In the hot plate test an increase of the reaction time was observed only at 60 (Ev=18.0+/-2.2; Em=20.8+/-2.52) and 90 min (Ev=20.4+/-1.71; Em=23.7+/-2.32) after the treatment with E. velutina and E. mulungu at the dose of 400 mg/kg as compared to controls (T60=11.1+/-0.74; T90=11.9+/-0.86). This effect was not reversed by naloxone. We conclude that E. velutina and E. mulungu presents antinociceptive effects, which are independent of the opioid system. (+info)