One-pot lipase-catalyzed esterification of ε-caprolactone with methyl-d-glucopyranoside and its elongation with free 6-hydroxyhexanoate monomer units.

One-pot synthesis of sugar-functionalized oligomeric caprolactone was carried out by lipase-catalyzed esterification of ε-caprolactone (ECL) with methyl-d-glucopyranoside (MGP) followed by the elongation of functionalized oligomer chain. Functionalization was performed in a custom-fabricated glass reactor equipped with Rushton turbine impeller and controlled temperature at 60 °C using tert-butanol as reaction medium. The overall reaction steps include MGP esterification of ECL monomer and its subsequent elongation by free 6-hydroxyhexanoate monomer units. A ping-pong bi-bi mechanism without ternary complex was proposed for esterification of ECL and MGP with apparent values of kinetic constant, namely maximal velocity (Vmax ), Michaelis constant for MGP (KmMGP ), and Michaelis constant for ECL (KmECL ) at 3.848 × 10-3  M H-1 , 8.189 × 10-2  M, and 6.050 M, respectively. Chain propagation step of MGP-functionalized ECL oligomer exhibits the properties of living polymerization mechanism. Linear relationship between conversion (%) and number average molecular weight, Mn (g mol-1 ), of functionalized oligomer was observed. Synthesized functionalized oligomer showed narrow range of molecular weight from 1,400 to 1,600 g mol-1 with more than 90% conversion achieved. Structural analysis confirmed the presence of covalent bond between the hydroxyl group in MGP with carboxyl end group of ECL oligomer.

A Galactoside-Binding Protein Tricked into Binding Unnatural Pyranose Derivatives: 3-Deoxy-3-Methyl-Gulosides Selectively Inhibit Galectin-1.

Galectins are a family of galactoside-recognizing proteins involved in different galectin-subtype-specific inflammatory and tumor-promoting processes, which motivates the development of inhibitors that are more selective galectin inhibitors than natural ligand fragments. Here, we describe the synthesis and evaluation of 3-C-methyl-gulopyranoside derivatives and their evaluation as galectin inhibitors. Methyl 3-deoxy-3-C-(hydroxymethyl)-ß-d-gulopyranoside showed 7-fold better affinity for galectin-1 than the natural monosaccharide fragment analog methyl ß-d-galactopyranoside, as well as a high selectivity over galectin-2, 3, 4, 7, 8, and 9. Derivatization of the 3-C-hydroxymethyl into amides gave gulosides with improved selectivities and affinities; methyl 3-deoxy-3-C-(methyl-2,3,4,5,6-pentafluorobenzamide)-ß-d-gulopyranoside had Kd 700 µM for galectin-1, while not binding any other galectin.

Conformational interchange of a carbohydrate by mechanical compression at the air-water interface.

Methyl xylopyranoside containing three 4-(pyrene-1-yl)benzoyl groups (PyXy) undergoes conformational interchange within a Langmuir monolayer upon mechanical compression. This xylose-type molecular machine PyXy was immobilized within two different matrix lipids, methyl stearate and methyl 2,3,4-tri-O-stearoyl-ß-D-xylopyranoside, which respectively form rigid and soft monolayers. Structural properties of the monolayer were characterized by assessing the compressibility, compression modulus, and ideal limiting molecular area of PyXy, all of which were estimated from the π-A isotherm measurements. Only the rigid monolayer exhibited a transition to the condensed phase with a limiting molecular area of PyXy smaller than that of the cross-sectional area of the xylopyranose ring in its C1 chair conformation. This suggests conformational interchange of PyXy from the most stable (4)C1 (C1) form to the metastable (1)C4 (1C) form. Surface-reflective fluorescence spectroscopy of the monolayer was applied to detect excimer emission resulting from the face-to-face dimerization of pyrenes attached at the O-2 and O-4 positions of xylose. Fluorescence intensity of the excimer increased abruptly in the condensed region only when the rigid monolayer was applied. These results indicate that the rigidity of the matrix monolayer is a critical aspect of the precise manipulation of molecular machines at interfaces. Consequently, this study demonstrates that including a molecular machine into a rigid lipid matrix is a promising means for the preparation of a novel nanoassembly with dynamic functionalities variable depending on a mechanical stimulus.

MBIP promotes ESCC metastasis by activating MAPK pathway.

MBIP is a component of the Ada2A containing complex (ATAC) and has been identified as a susceptibility gene in several cancers. However, the role and molecular mechanism of MBIP in esophageal squamous cell carcinoma (ESCC) remain unclear. Our finding indicated that the expression level of MBIP in ESCC was higher than that in normal tissue (P < 0.05) based on the data from the Cancer Gene Atlas (TCGA) and Gene Expression Omnibus (GEO). Kaplan-Meier analysis showed that high MBIP expression was closely associated with deeper invasion and worse prognosis. Transwell assay and mouse xenograft assay demonstrated that MBIP overexpression promoted migration and invasion in vitro and in vivo, while MBIP knockdown played the opposite role. Furthermore, the results of RNA-seq, qRT-PCR, western blotting and rescue experiments revealed that MBIP promoted epithelial-mesenchymal transition (EMT) via the phosphorylation JNK/p38 in ESCC. Our study indicates that MBIP plays a significant role in the prognosis and metastasis of ESCC, suggesting that MBIP might serve as an ESCC prognostic biomarker.

Carbohydrate-lipid interactions: affinities of methylmannose polysaccharides for lipids in aqueous solution.

The interactions between 3-O-methyl-mannose polysaccharides (MMPs), extracted from Mycobacterium smegmatis (consisting of a mixture of MMP-10, -11, -12 and -13) or obtained by chemical synthesis (MMP-5(s), -8(s), -11(s) and -14(s)), and linear saturated and unsaturated fatty acids (FAs), and a commercial mixture of naphthenic acids (NAs) in aqueous solution at 25 °C and pH 8.5 were quantified by electrospray ionization mass spectrometry (ESI-MS). Association constants (K(a)) for MMP binding to four FAs (myristic acid, palmitic acid, stearic acid and trans-parinaric acid) were measured by using an indirect ESI-MS assay, the "proxy protein" method. The K(a) values are in the 10(4)-10(5) M(-1) range and, based on results obtained for the binding of the synthetic MMPs with palmitic acid, increase with the size of the carbohydrate. Notably, the measured affinity of the extracted MMPs for trans-parinaric acid is two orders of magnitude smaller than the reported value, which was determined by using a fluorescence assay. Using a newly developed competitive binding assay, referred to as the "proxy protein/proxy ligand" ESI-MS method, it was shown that MMPs bind specifically to NAs in aqueous solution, with apparent affinities of approximately (5×10(4)) M(-1) for the mixture of NAs tested. This represents the first demonstration that MMPs can bind to hydrophobic species more complex than those containing linear alkyl/alkenyl chains. Moreover, the approach developed here represents a novel method for probing carbohydrate-lipid interactions.

Too low to kill: concentration of the secondary metabolite ranunculin in buttercup pollen does not affect bee larval survival.

Growing evidence suggests that the freely accessible pollen of some plants is chemically protected against pollen-feeding flower visitors. For example, a diet of pollen from buttercup plants (Ranunculus) recently was shown to have a deleterious effect on developing larvae of several bee species not specialized on Ranunculus. Numerous Ranunculus species contain ranunculin, the glucosyl hydrate form of the highly reactive and toxic lactone protoanemonin, that causes the toxicity of these plants. We tested whether the presence of ranunculin is responsible for the lethal effects of R. acris pollen on the larvae of two bee species that are not Ranunculus specialists. To investigate the effect on bee larval development, we added ranunculin to the pollen provisions of the Campanula specialist bee Chelostoma rapunculi and the Asteraceae specialist bee Heriades truncorum, and allowed the larvae to feed on these provisions. We quantified ranunculin in pollen of R. acris and in brood cell provisions collected by the Ranunculus specialist bee Chelostoma florisomne. We demonstrated that although ranunculin was lethal to both tested bee species in high concentrations, the concentration in the pollen of R. acris was at least fourfold lower than that tolerated by the larvae of C. rapunculi and H. truncorum in the feeding experiments. Ranunculin concentration in the brood cells of C. florisomne was on average even twentyfold lower than that in Ranunculus pollen, suggesting that a mechanism different from ranunculin intoxication accounts for the larval mortality reported for bees not specialized on Ranunculus pollen.

Methyl ß-lactoside [methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside] monohydrate: a solvomorphism study.

Methyl ß-lactoside [methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside] monohydrate, C13H24O11·H2O, (I), was obtained via spontaneous transformation of methyl ß-lactoside methanol solvate, (II), during air-drying. Cremer-Pople puckering parameters indicate that the ß-D-Galp (ß-D-galactopyranosyl) and ß-D-Glcp (ß-D-glucopyranosyl) rings in (I) adopt slightly distorted 4C1 chair conformations, with the former distorted towards a boat form (BC1,C4) and the latter towards a twist-boat form (O5SC2). Puckering parameters for (I) and (II) indicate that the conformation of the ßGalp ring is slightly more affected than the ßGlcp ring by the solvomorphism. Conformations of the terminal O-glycosidic linkages in (I) and (II) are virtually identical, whereas those of the internal O-glycosidic linkage show torsion-angle changes of 6° in both C-O bonds. The exocyclic hydroxymethyl group in the ßGalp residue adopts a gt conformation (C4' anti to O6') in both (I) and (II), whereas that in the ßGlcp residue adopts a gg (gauche-gauche) conformation (H5 anti to O6) in (II) and a gt (gauche-trans) conformation (C4 anti to O6) in (I). The latter conformational change is critical to the solvomorphism in that it allows water to participate in three hydrogen bonds in (I) as opposed to only two hydrogen bonds in (II), potentially producing a more energetically stable structure for (I) than for (II). Visual inspection of the crystalline lattice of (II) reveals channels in which methanol solvent resides and through which solvent might exchange during solvomorphism. These channels are less apparent in the crystalline lattice of (I).

Methyl beta-D-fructopyranoside.

In methyl beta-D-fructopyranoside, C(7)H(14)O(6), the thermodynamically most stable methyl glycoside of the ketose D-fructose, the pyranose ring is close to being an ideal (2)C(5) chair. The compound forms bilayers involving a complex hydrogen-bonding pattern of five independent hydrogen bonds. Graph-set analysis was applied to distinguish the hydrogen-bond patterns at unary and higher level graph sets.

Methyl 4-O-beta-D-galactopyranosyl alpha-D-mannopyranoside methanol 0.375-solvate.

Methyl beta-D-galactopyranosyl-(1-->4)-alpha-D-mannopyranoside methanol 0.375-solvate, C(13)H(24)O(11).0.375CH(3)OH, (I), was crystallized from a methanol-ethanol solvent system in a glycosidic linkage conformation, with varphi' (O5(Gal)-C1(Gal)-O1(Gal)-C4(Man)) = -68.2 (3) degrees and psi' (C1(Gal)-O1(Gal)-C4(Man)-C5(Man)) = -123.9 (2) degrees , where the ring is defined by atoms O5/C1-C5 (monosaccharide numbering); C1 denotes the anomeric C atom and C6 the exocyclic hydroxymethyl C atom in the betaGalp and alphaManp residues, respectively. The linkage conformation in (I) differs from that in crystalline methyl alpha-lactoside [methyl beta-D-galactopyranosyl-(1-->4)-alpha-D-glucopyranoside], (II) [Pan, Noll & Serianni (2005). Acta Cryst. C61, o674-o677], where varphi' is -93.6 degrees and psi' is -144.8 degrees . An intermolecular hydrogen bond exists between O3(Man) and O5(Gal) in (I), similar to that between O3(Glc) and O5(Gal) in (II). The structures of (I) and (II) are also compared with those of their constituent residues, viz. methyl alpha-D-mannopyranoside, methyl alpha-D-glucopyranoside and methyl beta-D-galactopyranoside, revealing significant differences in the Cremer-Pople puckering parameters, exocyclic hydroxymethyl group conformations and intermolecular hydrogen-bonding patterns.

Molecular specification and patterning of progenitor cells in the lateral and medial ganglionic eminences.

We characterized intrinsic and extrinsic specification of progenitors in the lateral and medial ganglionic eminences (LGE and MGE). We identified seven genes whose expression is enriched or restricted in either the LGE [biregional cell adhesion molecule-related/downregulated by oncogenes binding protein (Boc), Frizzled homolog 8 (Fzd8), Ankrd43 (ankyrin repeat domain-containing protein 43), and Ikzf1 (Ikaros family zinc finger 1)] or MGE [Map3k12 binding inhibitory protein 1 (Mbip); zinc-finger, SWIM domain containing 5 (Zswim5); and Adamts5 [a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5]]. Boc, Fzd8, Mbip, and Zswim5 are apparently expressed in LGE or MGE progenitors, whereas the remaining three are seen in the postmitotic mantle zone. Relative expression levels are altered and regional distinctions are lost for each gene in LGE or MGE cells propagated as neurospheres, indicating that these newly identified molecular characteristics of LGE or MGE progenitors depend on forebrain signals not available in the neurosphere assay. Analyses of Pax6(Sey/Sey), Shh(-/-), and Gli3(XtJ/XtJ) mutants suggests that LGE and MGE progenitor identity does not rely exclusively on previously established forebrain-intrinsic patterning mechanisms. Among a limited number of additional potential patterning mechanisms, we found that extrinsic signals from the frontonasal mesenchyme are essential for Shh- and Fgf8-dependent regulation of LGE and MGE genes. Thus, extrinsic and intrinsic forebrain patterning mechanisms cooperate to establish LGE and MGE progenitor identity, and presumably their capacities to generate distinct classes of neuronal progeny.

The major surface carbohydrates of the Echinococcus granulosus cyst: mucin-type O-glycans decorated by novel galactose-based structures.

The cestodes constitute important but understudied human and veterinary parasites. Their surfaces are rich in carbohydrates, on which very little structural information is available. The tissue-dwelling larva (hydatid cyst) of the cestode Echinococcus granulosus is outwardly protected by a massive layer of carbohydrate-rich extracellular matrix, termed the laminated layer. The monosaccharide composition of this layer suggests that its major carbohydrate components are exclusively mucin-type O-glycans. We have purified these glycans after their release from the crude laminated layer and obtained by MS and NMR the complete structure of 10 of the most abundant components. The structures, between two and six residues in length, encompass a limited number of biosynthetic motifs. The mucin cores 1 and 2 are either nondecorated or elongated by a chain of Galpbeta1-3 residues. This chain can be capped by a single Galpalpha1-4 residue, such capping becoming more dominant with increasing chain size. In addition, the core 2 N-acetylglucosamine residue is in cases substituted with the disaccharide Galpalpha1-4Galpbeta1-4, giving rise to the blood P(1)-antigen motif. Larger, also related, glycans exist, reaching at least 18 residues in size. The glycans described are related but larger than those previously described from an Echinococcus multilocularis mucin [Hulsmeier, A. J., et al. (2002) J. Biol. Chem. 277, 5742-5748]. Our results reveal that the E. granulosus cyst exposes to the host only a few different major carbohydrate motifs. These motifs are composed essentially of galactose units and include the elongation by (Galpbeta1-3)(n) and the capping by Galpalpha1-4, novel in animal mucin-type O-glycans.

The kinetics of cellular commitment during stimulation of lymphocytes by lectins.

The kinetics of cellular commitment in the stimulation of lymphocytes by concanavalin A (Con A) has been analyzed by measurement of DNA synthesis, autoradiography, and histologic staining techniques. If the competitive inhibitor alpha-methyl-D-mannoside (alphaMM) is introduced into cultures of mouse spleen cells at various times after the addition of Con A, there is a gradual decrease in its capacity to inhibit the lectin-stimulated incorporation of [(3)H]thymidine. Addition of the saccharide 20 h after exposure of the cells to Con A had no effect on the level of the cellular response to the lectin. With increasing periods of contact with Con A, the percentage of blast cells and the percentage of [(3)H]thymidine-labeled blast cells increased in parallel with the total radioactive thymidine incorporated while the average number of autoradiographic grains per labeled blast cell remained relatively constant. These observations suggest that the rising level of [(3)H]thymidine incorporation results from an increase in the number of cells that respond to lectin stimulation and become refractory to inhibition with alphaMM. Once such cells become committed, they synthesize DNA at a rate independent of the length of exposure to the lectin. The combined results indicate that mouse splenic lymphocytes are heterogeneous in their capacities to respond to Con A and that different cells require different induction periods to be stimulated.

Mitogen-stimulated glucose transport in thymocytes. Possible role of Ca++ and antagonism by adenosine 3':5'-monophosphate.

The plant lectin, concanavalin A (Con-A), and the ionophore, A-23187 (specific for divalent cations), stimulated glucose transport in rat thymocytes. Con-A stimulation developed more slowly and was somewhat less extensive than that of stimulation developed more slowly and was somewhat less extensive than that of A-23187. Both responses showed saturation dose dependencies. The two responses were poorly additive, suggesting that A-23187 may saturate regulatory processes shared by the two stimulatory mechanisms. Doses of methylisobutylxanthine (MIX) and prostaglandin E2 which raised adenosine 3':5'-monophosphate (cAMP) levels in these cells also antagonized the Con-A stimulation of glucose transport but did not inhibit basal glucose transport or the A-23187 stimulation. Dibutyryl-cAMP and 8-bromo-cAMP also natagonized Con-A stimulation without inhibiting basal glucose transport. MIX antagonized high Con-A doses about as strongly as it did low Con-A doses, suggesting that MIX did not compete in the Con-A binding step or other process saturable by Con-A. [3H-A1Con-A binding was not affected by MIX. The stimulatory effects of Con-A and A-23187 were reduced by reduction of Ca++ in the medium. Both Con-A and A-23187 enhanced 45Ca++ influx and cellular Ca++ content. The A-23187 dose, which was saturating for glucose transport stimulation, enhanced Ca++ influx and cellular Ca++ content more than did the Con-A dose which was saturating for glucose transport stimulation. The dose fo MIX which specifically antagonized Con-A stimulation of glucose transport proved also to reduce Ca++ influx and cellular Ca++ in the presence of Con-A but not in the presence of A-23187. Thus, glucose transport correlates rather well with cellular Ca++. These results are compatible with the view that Ca++ in a cellular compartment can promote glucose transport, the Con-A's enhancement of Ca++ entry contributes to its stimulation of glucose transport, and the MIX antagonized Con-A action at least partly by reducing Ca++ entry. The action of MIX is apparently mediated by cAMP.

Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells.

Schwann cells synthesize two heparan sulfate proteoglycans, one that is a component of the Schwann cell basement membrane and a smaller one that is an integral component of the Schwann cell plasma membrane. To determine the functions of these molecules, Schwann cell-nerve cell cultures were grown in medium containing a specific inhibitor of proteoglycan biosynthesis, 4-methylumbelliferyl-beta-D-xyloside. Treatment with 1 mM beta-D-xyloside caused a 90% reduction in the accumulation of 35SO4-labeled proteoglycans in the cell layer of the cultures. Gel filtration analysis revealed that both the basement membrane and plasma membrane proteoglycans were affected. Inhibition of proteoglycan biosynthesis was accompanied by an inhibition of laminin deposition into extracellular matrix as determined by immunostaining of cultures and by immunoblotting of cell-associated proteins. This occurred even though there was no decrease in the amount of laminin detected in the medium of beta-D-xyloside-treated cultures. Deposition of collagen type IV was similarly affected. In addition, there was no myelin produced in beta-D-xyloside treated cultures. However, when beta-xyloside-treated cultures were supplied with exogenous basement membrane, Schwann cells produced numerous myelin segments. These results indicate that Schwann cell proteoglycans play an essential role in basement membrane assembly, and that the integral plasma membrane proteoglycan is not required for the basement membrane to exert its effects on Schwann cell differentiation.

Concanavalin A increases the strength of baby hamster kidney cell attachment to substratum.

The strength of attachment of normal and transformed baby hamster kidney cells was markedly increased when attached cells were treated with concanavalin A (Con A). The cells became less sensitive to detachment by physical shear or by treatment with trypsin or EDTA; however, their morphology, as observed by phase contrast microscopy, did not change. The effects of Con A were prevented by the simultaneous addition of either D-glucose or alpha-methyl-D-glucoside with the Con A. Also addition of these reagents to the attached cells after Con A treatment partially reversed the effects caused by Con A. Pre-treatment of the culture flasks with Con A before cell attachment resulted in an increase in the strength of cell attachment to the culture flasks as compared to untreated controls.

Anomeric specificty of 3-0-methyl-D-glycopyranose against alloxan diabetes.

The individual alpha and beta anomers of the nonmetabolized glucose analog 3-O-methyl-D-glucopyranose (3MG) were studied as protective agents against the alloxan toxicity to pancreatic beta cells in an in vivo rat model. The alpha 3MG provides greater protection than either the beta or the equilibrated compound, as indicated by plasma glucose concentrations 24 hours after the experiment. This specificity suggests that the beta cell membrane is extremely stereospecific, and that glucose or 3MG provide protection against alloxan injury directly by an interaction with the cell membrane and not by subsequent metabolism of the protecting compound.

Concanavalin A: its action in inducing Oocyte maturation-inducing substance in starfish follicle cells.

Maturation of starfish oocytes is induced by 1-methyladenine produced in follicle cells under the influence of a gonad-stimulating hormonal peptide released from the nervous system. Concanavalin A stimulates isolated follicle cells to produce 1-methyladenine, which suggests that it has the same capacity as the peptide hormone to indirectly induce oocyte maturation.

Regulation of amino acid transport in kidney cortex of newborn rats.

After incubation at 37 degrees C the subsequent uptake of alpha-aminoisobutyric acid, cycloleucine, glycine, and L-proline by newborn (as compared to adult) rat kidney cortex slices is enhanced. The effect is abolished by the presence of cycloheximide, actinomycin D, and high concentrations of the above-mentioned amino acids in the medium during the 37 degrees C incubation prior to measurement of uptake. The data suggest that there is an adaptive control mechanism which is expressed on incubation at 37 degrees C and which can regulate amino acid transport in newborn rat kidney cortex.

Methyl 4,6-dichloro-4,6-dideoxy-alpha-D-galactopyranoside: an inhibitor of sweet taste responses in gerbils.

The sugar methyl 4,6-dichloro-4,6-dideoxy-alpha-D-galactopyranoside (DiCl-gal) is a new type of inhibitor of the gerbil's electrophysiological taste response to sucrose or saccharin. Saturated solutions of this compound alone barely stimulate the gerbil's taste nerve. But, when mixed with sucrose or saccharin, DiCl-gal suppresses the gerbil's taste response to these two sweeteners. In contrast, when mixed with sodium chloride or hydrochloric acid, DiCl-gal does not affect the taste responses to these compounds. However, unlike other inhibitors of sweet taste, the DiCl-gal taste suppression is short-lived and occurs only when the inhibitor is combined with the sweetener.

Synthesis of and molecular dynamics simulations on a tetrasaccharide corresponding to the repeating unit of the capsular polysaccharide from Salmonella enteritidis.

Syntheses of two oligosaccharides as methyl glycosides related to the repeating unit of S. enteritidis capsular polysaccharide (CPS) are presented. The trisaccharide corresponds to the backbone of the CPS whereas the tetrasaccharide is a model for the repeating unit which has a branched structure. Molecular dynamics simulations investigating their flexibility and dynamics revealed that the oligosaccharides populate several conformational states and indicate that conformational averaging should be used in describing the accessible conformational space.