Benildo Sousa Cavada

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) widespread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications.

Revisiting proteus: Do Minor Changes in Lectin Structure Matter in Biological Activity? Lessons from and Potential Biotechnological Uses of the Diocleinae Subtribe Lectins

Significant differences in function have been observed among lectins structurally similar to concanavalin A, but their high homology with this widely used lectin has kept them in obscurity. The observation of large differences in the potency of many of these Diocleinae lectins as stimulators of Interferon-g production by human peripheral blood mononuclear cells has lead to a major effort to unravel their chemical structure and biological activity. Modeling studies of some of these lectins reveal conformational changes in side chains of some residues involved in the carbohydrate-binding site, with possible effects on the ability of these proteins to recognize specific carbohydrate structures. Additionally, all them constitute in fact a mixture of isolectins, which in different proportions could lead to diverse effects. The present review of the biological actions of Diocleinae lectins includes several in vitro and in vivo immunological findings, as well as their effects on insect growth and reproduction. In these systems Diocleinae lectins proved to be quite diverse in their potency. Such diversity in the biological activity of highly related proteins recalls the origin of the name protein: like Proteus, the capability of assuming various forms is the essential feature of this class of molecules.

Compositional and nutritional attributes of seeds from the multiple purpose tree Moringa oleifera Lamarck

Moringa oleifera Lam is a multipurpose tree cultivated to use as a vegetable, for spice, for cooking and cosmetic oil and as a medicinal plant. Owing to the use of its seeds as food and as a clarifying agent of turbid water some nutritional and antinutritional characteristics were studied. The mature seeds contained 332.5 g crude protein, 412.0 g crude fat, 211.2 g carbohydrate and 44.3 g ash per kg dry matter. The essential amino acid profile compared with the FAO/WHO/UNU scoring pattern requirements for different age groups showed deficiency of lysine, threonine and valine. The content of methionine + cysteine (43.6 g kg−1 protein), however, was exceptionally higher and close to that of human milk, chicken egg and cows milk. The seed extract agglutinated rabbit erythrocytes but did not show trypsin inhibitor and urease activities. Feeding rats with a diet containing the seed meal showed loss of appetite, impaired growth, lower NPU and enlargement of stomach, small intestine, caecum + colon, liver, pancreas, kidneys, heart and lungs and atrophy of thymus and spleen in comparison with rats fed on an egg-white diet. The results indicated that consumption of M oleifera raw mature seeds should be viewed with some caution until suitable processing methods are developed to abolish the yet unknown adverse factors.

The crystal structure of Canavalia brasiliensis lectin suggests a correlation between its quaternary conformation and its distinct biological properties from Concanavalin A.

© 1997 Federation of European Biochemical Societies.

Rat paw edema and leukocyte immigration induced by plant lectins

Lectins fromDioclea grandiflora (DG) andCanavalia brasiliensis (CB) were compared with Concanavalin A (ConA) for their ability to induce paw edema and peritoneal cell immigration in rats. ConA caused a slight edema with a peak at 1 h after injection, while DG or CB induced a pronounced and long-lasting edema that reached a maximum at about 6 h. Different antiinflammatory drugs partially inhibited the edema. α-d-glucose (GLU) partially blocked the edema caused by ConA and markedly inhibited that due to CB, but had no effect on the edema induced by DG. α-Methyl mannoside (α-MM) blocked the edema caused by DG and ConA, but did not affect that caused by CB. At doses much lower than those used to induce paw edema, the lectins promoted an intense accumulation of neutrophil and mononuclear cells in the rat peritoneal cavity. CB and DG were more potent than ConA, which also presented a different profile of cell immigration. GLU significantly inhibited leukocyte accumulation caused by all lectins. α-MM impaired ConA- and DG-induced cell immigration, but only partially inhibited CB. Thus, despite their physicochemical similarities with ConA, DG and CB have more powerful pro-inflammatory effects. This difference seems to be related to their sugar-binding properties. However, while ConA- and DG-induced effects were inhibited more by α-MM than by GLU, CB-induced effects were inhibited more by glucose.

Binding Studies of α-GalNAc-specific Lectins to the α-GalNAc (Tn-antigen) Form of Porcine Submaxillary Mucin and Its Smaller Fragments

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.

Human Lymphocyte Stimulation by Legume Lectins from the Diocleae Tribe

Lectins from eight leguminous seeds from the Diocleae tribe were compared to Concanavalin A (Con A), a well known T cell mitogen, on the stimulation of lymphocyte proliferation and Interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) from normal volunteers. Lectins from Canavalia brasiliensis and Dioclea virgata induced the highest lymphocyte proliferation, both much higher than levels obtained with Con A, whereas lectins from Dioclea guianensis var. lasiophylla and from Canavalia bonariensis induced the lowest stimulation. Lectins from Dioclea rostrata, D. grandiflora, D. violacea and Cratylia floribunda induced intermediate levels of proliferation. The highest stimulation for IFN-gamma production was obtained with the lectin from D. rostrata, followed by those of C. floribunda and C. brasiliensis; only the lectins from D. virgata and C. bonariensis induced an IFN-gamma production lower than the one obtained by Con A-stimulation. Since all these legumes belong to the same tribe of C. ensiformis (Con A), and all are supposed to exhibit very similar lectins, it is interesting the high variation in the stimulation of lymphocyte proliferation. It is also noteworthy the dissociation between this parameter and IFN-gamma production in the case of D. virgata. A detailed analysis on the structure of such lectins, and their ligand sugars on lymphocyte surface is necessary to further explore such differences.

MOLECULAR CHARACTERIZATION AND CRYSTALLIZATION OF DIOCLEINAE LECTINS

Molecular characterization of seven Diocleinae lectins was assessed by sequence analysis, determination of molecular masses by mass spectrometry, and analytical ultracentrifugation equilibrium sedimentation. The lectins show distinct pH-dependent dimer-tetramer equilibria, which we hypothesize are due to small primary structure differences at key positions. Lectins from Dioclea guianensis, Dioclea virgata, and Cratylia floribunda seeds have been crystallized and preliminary X-ray diffraction analyses are reported.

Diocleinae Lectins Are a Group of Proteins with Conserved Binding Sites for the Core Trimannoside of Asparagine-linked Oligosaccharides and Differential Specificities for Complex Carbohydrates*

The seed lectin from Dioclea grandiflora and jack bean lectin concanavalin A (ConA) are both members of the Diocleinae subtribe of Leguminosae lectins. Both lectins have recently been shown to possess enhanced affinities and extended binding sites for the trisaccharide, 3,6-di-O-(α-d-mannopyranosyl)-d-mannose, which is present in the core region of all asparagine-linked carbohydrates (Gupta, D., Oscarson, S., Raju, S., Stanley, P. Toone, E. J. and Brewer, C. F. (1996) Eur. J. Biochem.242, 320–326). In the present study, the binding specificities of seven other lectins from the Diocleinae subtribe have been investigated by hemagglutination inhibition and isothermal titration microcalorimetry (ITC). The lectins are from Canavalia brasiliensis, Canavalia bonariensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. Hemagglutination inhibition and ITC experiments show that all seven lectins are Man/Glc-specific and have high affinities for the core trimannoside, like ConA and D. grandifloralectin. All seven lectins also exhibit the same pattern of binding to a series of monodeoxy analogs and a tetradeoxy analog of the trimannoside, similar to that of ConA and D. grandifloralectin. However, C. bonariensis, C. floribunda,D. rostrata, and D. violacea, like D. grandiflora, show substantially reduced affinities for a biantennary complex carbohydrate with terminal GlcNAc residues, whileC. brasiliensis, D. guianensis, and D. virgata, like ConA, exhibit affinities for the oligosaccharide comparable with that of the trimannoside. Thermodynamic data obtained by ITC indicate different energetic mechanisms of binding of the above two groups of lectins to the complex carbohydrate. The ability of the lectins to induce histamine release from rat peritoneal mast cells is shown to correlate with the relative affinities of the proteins for the biantennary carbohydrate.

Larvicidal activity of lectins from Myracrodruon urundeuva on Aedes aegypti

Aedes aegypti transmits etiologic agents of yellow fever and dengue. Vaccine for dengue virus is not available and vector control is essential to minimize dengue incidence. This report deals with the larvicidal activity of lectins isolated from Myracrodruon urundeuva bark (MuBL) and heartwood (MuHL). The lectins were isolated by ammonium sulphate treatment of crude extracts followed by chromatography on chitin. MuBL and MuHL were evaluated by electrophoresis under native (PAGE) and denaturing conditions (SDS-PAGE). Carbohydrate specificity of lectins was evaluated by hemagglutinating activity (HA) inhibition assay using N-acetyl-d-glucosamine and by affinity chromatography on N-acetyl-D-glucosamine immobilized in agarose gel. Larvicidal activity against A. aegypti was investigated with the extracts, salt fractions and isolated lectins. MuBL and MuHL were characterized by PAGE as basic proteins of molecular masses of 14.0 and 14.4 kDa, respectively. The interaction of lectins with N-acetylglucosamine was detected by inhibition of HA by monosaccharide and lectin adsorptions on N-acetyl-D-glucosamine matrix. All M. urundeuva preparations promoted larvae mortality. LC16, LC50 and LC84 values of 0.077, 0.125, 0.173 for MuBL and 0.03, 0.04 and 0.05 mg/mL for MuHL were obtained. To our knowledge this is the first report of larvicidal activity of lectins against A. aegypti.