Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

Sweetening agents of plant origin: Phenylpropanoid constituents of seven sweet-tasting plants

Field inquiries and organoleptic tests for sweet taste led to the procurement of samples ofPiper marginatum (dried leaves),Tagetes filicifolia (fresh whole plants),Osmorhiza longistylis (fresh roots),Foeniculum vulgare (fresh aerial parts),Myrrhis odorata (fresh whole plants),Ocimum basilicum (fresh aerial parts), andIllicium verum (dried fruits). Follow-up laboratory studies of the leaves ofPiper marginatum demonstrated that trans-anethole (a phenylpropanoid) was the major sweet constituent of this species. In the remaining six species, GC/MS analysis also enabled us to demonstrate that sweetness is attributable, in each case, to the presence of high concentrations of the phenylpropanoids, trans-anethole and estragole, either alone or in combination.     

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts

Metschnikowia reukaufii is a widespread yeast able to grow in the plants’ floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l⁻¹, which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload-sucrose reactions. These products were purified and structurally characterized by MS-ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α-(1→1), α-(1→3) and α-(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l⁻¹, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l⁻¹ of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.     

Salicylic acid prevents Trichoderma harzianum from entering the vascular system of roots

Trichoderma is a soil‐borne fungal genus that includes species with a significant impact on agriculture and industrial processes. Some Trichoderma strains exert beneficial effects in plants through root colonization, although little is known about how this interaction takes place. To better understand this process, the root colonization of wild‐type Arabidopsis and the salicylic acid (SA)‐impaired mutant sid2 by a green fluorescent protein (GFP)‐marked Trichoderma harzianum strain was followed under confocal microscopy. Trichoderma harzianum GFP22 was able to penetrate the vascular tissue of the sid2 mutant because of the absence of callose deposition in the cell wall of root cells. In addition, a higher colonization of sid2 roots by GFP22 compared with that in Arabidopsis wild‐type roots was detected by real‐time polymerase chain reaction. These results, together with differences in the expression levels of plant defence genes in the roots of both interactions, support a key role for SA in Trichoderma early root colonization stages. We observed that, without the support of SA, plants were unable to prevent the arrival of the fungus in the vascular system and its spread into aerial parts, leading to later collapse.     

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.     

Efficiency of different egg parasitoids and increased floral diversity for the biological control of noctuid pests

Augmentative biological control of insects is a tool of Integrated Pest Management programs. In many agroecosystems, biological control is exercised largely by parasitoids, and it is found that the presence of food resources, as provided by flowering plants, can have a positive effect on survival, search ability and parasitism rate of parasitoid species. In Colombia, a recently established export crop, the cape gooseberry, is under continuous attack by a Lepidopteran species complex in the family Noctuidae. Our first objective was to test the longevity and parasitism rates of three species of egg parasitoids in the genus Trichogramma (Trichogramma atopovirilia Oatman & Platner, Trichogramma exiguum Pinto & Platner, and Trichogramma pretiosum Riley) for Spodoptera frugiperda Smith and Copitarsia decolora Gueené. Our results suggest that T. atopovirilia and T. pretiosum could be promising parasitoids for the control of S. frugiperda and C. decolora, with a percentage parasitism of between 30% and 60%, respectively. For our second objective, we selected T. atopovirilia as a model species to evaluate the effect of the presence of flowering plants on the longevity and parasitism rate in both, no choice and multiple choice experiments, under laboratory and field conditions. Our results consistently showed that the presence of red clover (Trifolium pratense L.) can increase the longevity and parasitism rate of T. atopovirilia, suggesting that providing food resources to parasitoids in cape gooseberry fields should be part of a habitat diversification strategy to control noctuid pests.     

A consensus ethnobotany of the Q’eqchi’ Maya of Southern Belize

An ethnobotany study in collaboration with Q’eqchi’ Maya healers of Southern Belize led to a collection of 169 medicinal plant species, belonging to 67 different families. The data show the use of a majority of species from primary or secondary semi-evergreen rainforests of Southern Belize, rather than weedy species. The medicinal uses of the plants were grouped into 17 usage categories. The frequency of use for each plant and the informant consensus factor for each usage category reveals a consensus among the healers on the use of plant species as well as on the diseases treated. These results suggest a well-defined medicinal tradition.     

N-type inactivation of the potassium channel KcsA by the Shaker B "ball" peptide: mapping the inactivating peptide-binding epitope

The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr(8), which exhibit the strongest STD effects; the C4H in the imidazole ring of His(16); the methyl protons of Val(4), Leu(7), and Leu(10) and the side chain amine protons of one, if not both, the Lys(18) and Lys(19) residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val(4) and Leu(10), whereas His(16) seems similarly affected as before. Conversely, STD effects on Tyr(8) are strongly diminished, and those on Lys(18) and/or Lys(19) are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of (13)C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a beta-hairpin structure when bound to the KcsA channel. Indeed, docking such a beta-hairpin structure into an open pore model for K(+) channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.