Renal alterations promoted by the lectins from Canavalia ensiformis (ConA) and Dioclea guianensis (DguiL) seeds

The lectin from the seeds of Canavalia ensiformis (ConA) and Dioclea guianensis (DguiL) was tested upon its renal effects using the isolated perfusion rat kidney method. Both lectins (10 microg/ml) affected perfusion pressure and renal vascular resistance, but DguiL showed a much greater action than ConA. However, ConA, but not DguiL, affected potassium tubular transport.     

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.      

Thermodynamic binding studies of lectins from the diocleinae subtribe to deoxy analogs of the core trimannoside of asparagine-linked oligosaccharides

Lectins from seven different species of the Diocleinae subtribe have been recently isolated and characterized in terms of their carbohydrate binding specificities (Dam, T. K., Cavada, B. S., Grangeiro, T. B., Santos, C. F., de Sousa, F. A. M., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 12082-12088). The lectins included those from Canavalia brasiliensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. All of the lectins exhibited specificity for Man and Glc residues, but much higher affinities for the branched chain trimannoside, 3,6-di-O-(alpha-d-mannopyranosyl)-d-mannose, which is found in the core region of all asparagine-linked carbohydrates. In the present study, isothermal titration microcalorimetry is used to determine the binding thermodynamics of the above lectins, including a new lectin from Canavalia grandiflora, to a complete series of monodeoxy analogs of the core trimannoside. From losses in the affinity constants and enthalpies of binding of certain deoxy analogs, assignments are made of the hydroxyl epitopes on the trimannoside that are involved in binding to the lectins. The pattern of binding of the deoxy analogs is similar for all seven lectins, and similar to that of concanavalin A which is also a member of the Diocleinae subtribe. However, differences in the magnitude of the thermodynamic binding parameters of the lectins are observed, even though the lectins possess conserved contact residues in many cases, and highly conserved primary sequences. The results indicate that non-contact residues in the lectins, even those distant from the binding sites, modulate their thermodynamic binding parameters.     

The amino-acid sequence of the glucose/mannose-specific lectin isolated from Parkia platycephala seeds reveals three tandemly arranged jacalin-related domains.

A mannose/glucose-specific lectin was isolated from seeds of Parkia platycephala, the most primitive subfamily of Leguminosae plants. The molecular mass of the purified lectin determined by mass spectrometry was 47 946 +/- 6 Da (by electrospray ionization) and 47 951 +/- 9 Da (by matrix-assisted laser-desoption ionization). The apparent molecular mass of the lectin in solutions of pH in the range 4.5-8.5 determined by analytical ultracentrifugation equilibrium sedimentation was 94 +/- 3 kDa, showing that the protein behaved as a non-pH-dependent dimer. The amino-acid sequence of the Parkia lectin was determined by Edman degradation of overlapping peptides. This is the first report of the primary structure of a Mimosoideae lectin. The protein contained a blocked N-terminus and a single, nonglycosylated polypeptide chain composed of three tandemly arranged homologous domains. Each of these domains shares sequence similarity with jacalin-related lectin monomers from Asteraceae, Convolvulaceae, Moraceae, Musaceae, Gramineae, and Fagaceae plant families. Based on this homology, we predict that each Parkia lectin repeat may display a beta prism fold similar to that observed in the crystal structure of the lectin from Helianthus tuberosus. The P. platycephala lectin also shows sequence similarity with stress- and pathogen-upregulated defence genes of a number of different plants, suggesting a common ancestry for jacalin-related lectins and inducible defence proteins.     

The interaction of Vatairea macrocarca and Rhizobium tropici: net H+ efflux stimulus and alteration of extracellular Na+ concentration

We studied the effect of a lectin isolated from seeds of the legume Vatairea macrocarpa on net H+ efflux in Rhizobium tropici, a bacterium capable of nodulating legume Phaseolus vulgaris. V. macrocarpa lectin (VML) was observed to temporarily stimulate the specific net H(+) efflux in R. tropici. When VML was present at 32 microg ml(-1), with or without 2 microM galactose (Gal), a specific net efflux >2.4 pM H+(min)(-1) mg dry biomass(-1) was induced. There was no detectable net H+ efflux when bovine serum albumin (16 microg ml(-1)) was tested. Addition of 16 microgVMLml(-1) resulted in a 700% increase of the extracellular Na+ concentration. The soluble proteins in the supernatant containing VML extract indicate a maximum immobilization of +/-10 microgVMLml(-1), with a minimum of 36,600 dimers or 8500 larger aggregates of VML binding in each bacterium. Our data suggest that VML activates Rhizobium as a bioenergetic substrate molecule, resulting in potential alterations of the external bacterial membrane.     

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.     

Primates in Human-Modified and Fragmented Landscapes: The Conservation Relevance of Modelling Habitat and Disturbance Factors in Density Estimation

Accurate density estimations of threatened animal populations is essential for management and conservation. This is particularly critical for species living in patchy and altered landscapes, as is the case for most tropical forest primates. In this study, we used a hierarchical modelling approach that incorporates the effect of environmental covariates on both the detection (i.e. observation) and the state (i.e. abundance) processes of distance sampling. We applied this method to already published data on three arboreal primates of the Udzungwa Mountains of Tanzania, including the endangered and endemic Udzungwa red colobus (Procolobus gordonorum). The area is a primate hotspot at continental level. Compared to previous, ‘canonical’ density estimates, we found that the inclusion of covariates in the modelling makes the inference process more informative, as it takes in full account the contrasting habitat and protection levels among forest blocks. The correction of density estimates for imperfect detection was especially critical where animal detectability was low. Relative to our approach, density was underestimated by the canonical distance sampling, particularly in the less protected forest. Group size had an effect on detectability, determining how the observation process varies depending on the socio-ecology of the target species. Lastly, as the inference on density is spatially-explicit to the scale of the covariates used in the modelling, we could confirm that primate densities are highest in low-to-mid elevations, where human disturbance tend to be greater, indicating a considerable resilience by target monkeys in disturbed habitats. However, the marked trend of lower densities in unprotected forests urgently calls for effective forest protection.     

Lectin from Canavalia brasiliensis (ConBr) protects hippocampal slices against glutamate neurotoxicity in a manner dependent of PI3K/Akt pathway

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 6 h 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, p38^MAPK 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 p38^MAPK 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.