Biomedical subjects
Publications and source records attributed to M G Hahn.
The structural element of alicyclic beta-amino acids shows some remarkable biological effects: For some 5- and 6-membered beta-amino acids a unique anti fungal activity has been observed, 7-membered beta-amino acid derivatives have been investigated for neurological disorders. The application of 5-, 6- and 7-membered alicyclic beta-amino acids in Medicinal Chemistry will be reported. [structure: see text]
A full account on a total synthesis of GPI anchor 1 employing butanediacetal (BDA) groups and a chiral bis(dihydropyran) is presented. The reactivity of selenium and thio glycosides was tuned by the use of BDA groups. This allowed the assembly of an appropriately protected GPI anchor precursor 2 in just six steps from the six building blocks 5-10 including only one protecting group manipulation. myo-Inositol was desymmetrised with the bis(dihydropyran) derivative 15 and appropriately protected to give inositol acceptor 21 in nine steps and 17% overall yield. The use of common starting materials and BDA-protections give efficient access to building blocks 5, 6, 7 and 8. A new and improved synthesis of the glucosamine donor 28 is included. In summary, a highly convergent and efficient synthesis of GPI anchor 1, which is clearly adaptable to other GPI anchors, has been reported.
Previous studies have led to the identification and characterization of specific, high-affinity binding sites for a hepta-beta-glucoside elicitor in soybean. A survey of plant species for elicitor-binding activity reveals that among the plants tested, the hepta-beta-glucoside elicitor is only recognized by plants belonging to the legume family. We have characterized in detail the glucan elicitor-binding site in the model legume Medicago truncatula Gaertn., and partially characterized the site in Lotus japonicus. These sites have characteristics that are very similar to the one in soybean, with dissociation constants of 4.7 and 8.9 nM respectively. The elicitor-binding sites from both plants are stable during solubilization with non-ionic alkylglycoside detergents. However, differences are observed in the abundance of the binding sites and their selectivity towards structurally related analogues of the hepta-beta-glucoside elicitor. Our results suggest that similar, but perhaps not identical, binding sites for the hepta-beta-glucoside elicitor exist in diverse legumes, but not in plants outside of the legume family.
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In the Arabidopsis root meristem, initial cells undergo asymmetric divisions to generate the cell lineages of the root. The scarecrow mutation results in roots that are missing one cell layer owing to the disruption of an asymmetric division that normally generates cortex and endodermis. Tissue-specific markers indicate that a heterogeneous cell type is formed in the mutant. The deduced amino acid sequence of SCARECROW (SCR) suggests that it is a member of a novel family of putative transcription factors. SCR is expressed in the cortex/endodermal initial cells and in the endodermal cell lineage. Tissue-specific expression is regulated at the transcriptional level. These results indicate a key role for SCR in regulating the radial organization of the root.
Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.
Elicitors are molecules that stimulate any of a number of defense responses in plants. Research over the past decade has focused on the mechanisms by which plant cells perceive and transduce these biological signals to activate defense responses. Of particular interest has been the identification of specific elicitor-binding proteins that might function as physiological receptors in the signal transduction cascade. The existence of specific high-affinity binding sites has been demonstrated for oligosaccharide, glycopeptide, and peptide elicitors, and candidate elicitor-binding proteins have been identified for several of them. The properties of these binding sites/proteins are consistent with those expected of physiologically important receptors, although experimental verification of the role of these binding proteins as receptors has not yet been obtained. The purification and characterization of specific elicitor-binding proteins is essential for a detailed understanding of the molecular basis for the signal exchange between plant hosts and microbial pathogens that leads to activation of host defenses.
Monoclonal antibody CCRC-M7 is representative of a group of antibodies with similar binding specificity that were generated using the plant cell-wall pectic polysaccharide, rhamnogalacturonan I, as immunogen. The epitope recognized by CCRC-M7 is present in several plant polysaccharides and membrane glycoproteins. Selective enzymatic or chemical removal of arabinosyl residues from rhamnogalacturonan I reduced, but did not abolish, the ability of CCRC-M7 to bind to the polysaccharide. In contrast, enzymatic removal of both arabinosyl and galactosyl residues from rhamnogalacturonan I completely abolished binding of CCRC-M7 to the resulting polysaccharide. Competitive ELISAs using chemically defined oligosaccharides to compete for the CCRC-M7 binding site showed that oligosaccharides containing (1-->6)-linked beta-D-galactosyl residues were the best competitors among those tested, with the tri-, penta-, and hexa-saccharides being equally effective. The combined results from indirect and competitive ELISAs suggest that the minimal epitope recognized by CCRC-M7 encompasses a (1-->6)-linked beta-galactan containing at least three galactosyl residues with at least one arabinosyl residue attached.
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Monoclonal antibodies (McAbs) generated against rhamnogalacturonan I (RG-I) purified from suspension-cultured sycamore maple (Acer pseudoplatanus) cells fall into three recognition groups. Four McAbs (group I) recognize an epitope that appears to be immunodominant and is present on RG-I from maize and sycamore maple, pectin and polygalacturonic acid from citrus, gum tragacanth, and membrane glycoproteins from suspension-cultured cells of maize, tobacco, parsley, bean, and sycamore maple. A second set of McAbs (group II) recognizes an epitope present in sycamore maple RG-I but does not bind to any of the other polysaccharides or glycoproteins recognized by group I. Lastly, one McAb, CCRC-M1 (group III), binds to RG-I and more strongly to xyloglucan (XG) from sycamore maple but not to maize RG-I, citrus polygalacturonic acid, or to the plant membrane glycoproteins recognized by group I. The epitope to which CCRC-M1 binds has been examined in detail. Ligand competition assays using a series of oligosaccharides derived from or related to sycamore maple XG demonstrated that a terminal alpha-(1-->2)-linked fucosyl residue constitutes an essential part of the epitope recognized by CCRC-M1. Oligosaccharides containing this structural motif compete with intact sycamore maple XG for binding to the antibody, whereas structurally related oligosaccharides, which do not contain terminal fucosyl residues or in which the terminal fucosyl residue is linked alpha-(1-->3) to the adjacent glycosyl residue, do not compete for the antibody binding site. The ligand binding assays also indicate that CCRC-M1 binds to a conformationally dependent structure of the polysaccharide. Other results of this study establish that some of the carbohydrate epitopes of the plant extracellular matrix are shared among different macromolecules.
We are studying the cellular signalling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-beta-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete, Phytophthora megasperma f. sp. glycinea. Our research has focused on the first step in this signal pathway, namely the specific recognition of the hepta-beta-glucoside elicitor by plasma-membrane-localized binding protein(s) in soybean cells. Binding of a radio-iodinated derivative of the elicitor-active hepta-beta-glucoside by membrane elicitor-binding proteins is specific, reversible, saturable and of high affinity (Kd = 0.75 nM). After solubilization using the non-ionic detergent n-dodecylsucrose, the elicitor-binding proteins retain the binding affinity (Kd = 1.8 nM) for the radiolabelled elicitor and the binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of elicitor-active and structurally related inactive oligoglucosides to displace labelled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-beta-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the elicitor-binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification and cloning of the hepta-beta-glucoside elicitor-binding proteins. Purification and characterization of the hepta-beta-glucoside-binding protein(s) or their corresponding cDNAs is a first step toward elucidating how the hepta-beta-glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.
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Plant and fungal cells are surrounded by a cell wall rich in diverse polysaccharides and proteins. It has become apparent in recent years that the carbohydrates in the cell wall function not only to maintain cell shape and integrity, but also may serve as signals in plants. This review summarizes the evidence that biologically-active oligosaccharides (oligosaccharins) released from plant or microbial cell walls can serve as signals to regulate plant defense and plant growth and development. The oligosaccharins discussed include the fungal-derived hepta-beta-glucoside and the plant cell wall-derived oligogalacturonides and xyloglucans. Possible mechanisms by which oligosaccharins may exert their effects on plant cells are discussed.
Total membranes prepared from roots of soybean (Glycine max L.) seedlings have previously been shown to contain proteinaceous binding site(s) for a hepta-beta-glucoside elicitor of phytoalexin accumulation. The hepta-beta-glucoside elicitor-binding proteins have now been shown to co-migrate with a plasma membrane marker enzyme (vanadate-sensitive H(+)-ATPase) on linear sucrose density gradients. With the use of detergents, the elicitor-binding proteins have been solubilized in functional form from soybean root membranes. The nonionic detergents n-dodecylsucrose, n-dodecylmaltoside, and Triton X-114, at concentrations of 5 to 10 mg/mL, each solubilizes between 50 and 60% of the elicitor-binding activity in a single extraction of the membranes. A zwitterionic detergent, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (ZW 3-12), also solubilizes about 40% of the total binding activity at detergent concentrations between 1 and 2 mg/mL, but the total binding activity recovered is only approximately 50% of that recovered with the nonionic detergents. The elicitor-binding proteins solubilized with either n-dodecylsucrose or ZW 3-12 retain the high affinity for radiolabeled hepta-beta-glucoside elicitor (apparent dissociation constant [Kd] = 1.8 nM and 1.4 nM, respectively) that was observed with the membrane-localized binding proteins (apparent Kd = 1 nM). Competitive ligand-binding experiments with several structurally related synthetic oligoglucosides demonstrate that the solubilized binding proteins retain specificity for elicitor-active oligosaccharides, irrespective of the detergent used for solubilization. Moreover, the binding affinities of the oligoglucosides for the solubilized binding proteins correlate well with their abilities to induce phytoalexin accumulation in soybean cotyledon tissue. Gel-permeation chromatography of n-dodecylsucrose-solubilized elicitor-binding proteins demonstrate that the bulk of the elicitor-binding activity is associated with large detergent-protein micelles (relative molecular weight > 400,000). Our results suggest that n-dodecylsucrose is a suitable detergent for solubilizing elicitor-binding proteins from soybean root membranes with minimal losses of binding activity. More importantly, we demonstrate that solubilization does not significantly after the binding properties of the proteins for elicitor-active oligoglucosides.
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