A new lectin in red kidney beans called PvFRIL stimulates proliferation of NIH 3T3 cells expressing the Flt3 receptor.

A new legume lectin has been identified by its ability to specifically stimulate proliferation of NIH 3T3 fibroblasts expressing the Flt3 tyrosine kinase receptor. The lectin was isolated from conditioned medium harvested from human peripheral blood mononuclear cells activated to secrete cytokines by a crude red kidney bean extract containing phytohemagglutinin (PHA). Untransfected 3T3 cells and 3T3 cells transfected with the related Fms tyrosine kinase receptor do not respond to this lectin, which we called PvFRIL (Phaseolus vulgaris Flt3 receptor-interacting lectin). When tested on cord blood mononuclear cells enriched for Flt3-expressing progenitors, purified PvFRIL fractions maintained a small population of cells that continued to express CD34 after 2 weeks in suspension cultures containing IL3. These cultures did not show the effects of IL3's strong induction of proliferation and differentiation (high cell number and exhausted medium); instead, low cell number at the end of the culture period resulted in persistence of cells in the context of cell death. These observations led to the hypothesis that PvFRIL acts in a dominant manner to preserve progenitor viability and prevent proliferation and differentiation.

The role of weak protein-protein interactions in multivalent lectin-carbohydrate binding: crystal structure of cross-linked FRIL.

Binding of multivalent glycoconjugates by lectins often leads to the formation of cross-linked complexes. Type I cross-links, which are one-dimensional, are formed by a divalent lectin and a divalent glycoconjugate. Type II cross-links, which are two or three-dimensional, occur when a lectin or glycoconjugate has a valence greater than two. Type II complexes are a source of additional specificity, since homogeneous type II complexes are formed in the presence of mixtures of lectins and glycoconjugates. This additional specificity is thought to become important when a lectin interacts with clusters of glycoconjugates, e.g. as is present on the cell surface. The cryst1al structure of the Glc/Man binding legume lectin FRIL in complex with a trisaccharide provides a molecular snapshot of how weak protein-protein interactions, which are not observed in solution, can become important when a cross-linked complex is formed. In solution, FRIL is a divalent dimer, but in the crystal FRIL forms a tetramer, which allows for the formation of an intricate type II cross-linked complex with the divalent trisaccharide. The dependence on weak protein-protein interactions can ensure that a specific type II cross-linked complex and its associated specificity can occur only under stringent conditions, which explains why lectins are often found forming higher-order oligomers.

Purification and characterization of Dolichos lablab lectin.

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin-Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.

cDNA cloning of FRIL, a lectin from Dolichos lablab, that preserves hematopoietic progenitors in suspension culture.

Ex vivo culture of hematopoietic stem cells is limited by the inability of cytokines to maintain primitive cells without inducing proliferation, differentiation, and subsequent loss of repopulating capacity. We identified recently in extracts of kidney bean and hyacinth bean a mannose-binding lectin, called FRIL, and provide here evidence that this protein appears to satisfy properties of a stem cell preservation factor. FRIL was first identified based on its ability to stimulate NIH 3T3 cells transfected with Flt3, a tyrosine kinase receptor central to regulation of stem cells. Molecular characterization from polypeptide sequencing and identification of the cDNA of hyacinth bean FRIL shows 78% amino acid identity with a mannose-binding lectin of hyacinth beans. Treatment of primitive hematopoietic progenitors in suspension culture with purified hyacinth FRIL alone is able to preserve cells for 1 month without medium changes. In vitro progenitor assays for human hematopoietic cells cultured 3 weeks in FRIL displayed small blast-like colonies that were capable of serial replating and persisted even in the presence of cytokines known to induce differentiation. These results suggest that FRIL is capable of preserving primitive progenitors in suspension culture for prolonged periods. FRIL's clinical utility involving procedures for stem cell transplantation, tumor cell purging before autologous transplantation, and ex vivo cultures used for expansion and stem cell gene therapy currently are being explored.

Olfactory, gas chromatographic and mass-spectral analyses of fecal volatiles traced to ingested licorice and apple.

Volatile components of two foodstuffs with characteristic aromas, apple and licorice, and fecal samples obtained from subjects on high-apple and licorice diets, were analyzed by head-space gas chromatography and gas chromatography-mass spectrometer. The volatile compounds characteristic of the odors of apple and licorice were detected by "sniffing" the GC effluent. The aromatic component of licorice was identified by GC-MS as anethole [1-methoxy-4-(2-propenyl) benzene]. The aromatic component of apple could not be chemically characterized by our GC-MS system even though readily detected by the nose. Head-space, GC-MS analysis of fecal volatiles offers a means of tracing odorous and non-odorous components to ingested foodstuffs with potential applications to clinical and forensic medicine and anthropology.

Dawn diarrhea.

A case of detergent-induced hemorrhagic colitis is presented. The endoscopic findings which were suggestive of a vasculitis, are depicted and discussed.