MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates.

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. The MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 µg mL-1 without interference from other algae acidic hydrolyzate components.

Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.

Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We now have developed this molecular strategy to render cytotoxic T cells specific for fungi. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ (designated "D-CAR") upon binding with carbohydrate in the cell wall of Aspergillus germlings. T cells genetically modified with the Sleeping Beauty system to express D-CAR stably were propagated selectively on artificial activating and propagating cells using an approach similar to that approved by the Food and Drug Administration for manufacturing CD19-specific CAR(+) T cells for clinical trials. The D-CAR(+) T cells exhibited specificity for ß-glucan which led to damage and inhibition of hyphal growth of Aspergillus in vitro and in vivo. Treatment of D-CAR(+) T cells with steroids did not compromise antifungal activity significantly. These data support the targeting of carbohydrate antigens by CAR(+) T cells and provide a clinically appealing strategy to enhance immunity for opportunistic fungal infections using T-cell gene therapy.

α-Amylase inhibitory activity from nut seed skin polyphenols. 1. Purification and characterization of almond seed skin polyphenols.

Using α-amylase inhibition as a separation guide, polyphenolic compounds from almond ( Prunus dulcis ) seed skin were purified using ultrafiltration and Sephadex LH-20 and ODS columns. The purified fraction specifically and strongly inhibited α-amylase; the IC50 value was 2.2 µg/mL for pig pancreatic α-amylase. The fraction contained about 62% of the total polyphenols, 33.8% flavanol-type tannins and 30% procyanidins. Oral administration of the polyphenol fraction to rats fed corn starch significantly suppressed an increase in blood glucose levels and area under the curve (AUC), in a dose-dependent manner. High-resolution MALDI-TOF mass spectra showed that the structure of this sample is a series of polyflavan-3-ol polymers composed of catechin/epicatechin units and gallocatechin/epigallocatechin units up to 11-mer with several interflavanoid ether linkages. The results suggest almond seed skin contains highly polymerized polyphenols with strong α-amylase inhibitory activity, which retard absorption of carbohydrate.

Presença de inibidores de proteases em amostras comerciais de “faseolaminas” utilizadas como bloqueadores de carboidratos e os riscos à saúde / Occurrence of proteases inhibitors in commercial samples of “phaseolamins” used as carbohydrate blockers and the health risks

Foi investigada a veiculação de inibidores de proteases, concomitante ao consumo de “faseolamina”, cuja ingestão diária e de maneira prolongada poderia estar associada ao risco no desenvolvimento de alterações morfológicas e metabólicas de pâncreas. Dez amostras foram obtidas em farmácias de manipulação no município de Uberaba – MG, as quais são comercializadas na forma de cápsulas como “faseolamina”e uma como “farinha de feijão branco”, além de amostra de farinha preparada a partir da trituração de feijões brancos (Phaseolus vulgaris) obtidos comercialmente, sem qualquer processamento. Estas amostras foram analisadas efetuando-se a determinação de atividade de inibidores de tripsina, a determinação de proteínas totais e de atividade de inibição de amilase. Todas as amostras estudadas apresentaram atividades de inibição de tripsina, porém com baixa ou mesmo inexistente inibição de amilase; e em algumas amostras foi detectada concentração de proteínas muito inferior ao esperado. Uma vez observada a presença de atividade de inibição de tripsina em todas as amostras estudadas, e pela inexistência de níveis seguros de consumo estabelecidos para o ser humano, e ainda baseando-se nos trabalhos sobre associação de ingestão prolongada destes inibidores com alterações de atividade pancreática, o consumo destes “bloqueadores de carboidratos” deveria ser melhor controlado.

This study aimed at verifying the risk in developing the metabolic and morphological changes inpancreas, owing to the use of daily and prolonged consumption of protease inhibitors in conjunctionwith “phaseolamine”. Ten samples were obtained from drugstores in the city of Uberaba – MG, soldas “phaseolamine” and one known as “white bean flour”, and also one flour sample produced fromcommercial white beans (Phaseolus vulgaris) without any processing. These samples were analyzedby determining the activity of trypsin inhibitors, the total protein and the amylase inhibition activity.Trypsin inhibition activity was found in all of analyzed samples; however, the amylase inhibition activitywas low, and in some samples the protein concentration was much lower than expected. Seeing that thetrypsin inhibitory activity was detected in all of analyzed samples, and considering that the prolongedingestion of these inhibitors might cause activity changes in pancreas, the consumption of these types of“carbohydrate blockers” should be deeply discussed, since the safe amounts of these compounds have notbeen established for humans consumption yet.

An array-based method to identify multivalent inhibitors.

Carbohydrate-protein interactions play a critical role in a variety of biological processes, and agonists/antagonists of these interactions are useful as biological probes and therapeutic agents. Most carbohydrate-binding proteins achieve tight binding through formation of a multivalent complex. Therefore, both ligand structure and presentation contribute to recognition. Since there are many potential combinations of structure, spacing, and orientation to consider and the optimal one cannot be predicted, high-throughput approaches for analyzing carbohydrate-protein interactions and designing inhibitors are appealing. In this report, we develop a strategy to vary neoglycoprotein density on a surface of a glycan array. This feature of presentation was combined with variations in glycan structure and glycan density to produce an array with approximately 600 combinations of glycan structure and presentation. The unique array platform allows one to distinguish between different types of multivalent complexes on the array surface. To illustrate the advantages of this format, it was used to rapidly identify multivalent probes for various lectins. The new array was first tested with several plant lectins, including concanavalin A (conA), Vicia villosa isolectin B4 (VVL-B(4)), and Ricinus communis agglutinin (RCA120). Next, it was used to rapidly identify potent multivalent inhibitors of Pseudomonas aeruginosa lectin I (PA-IL), a key protein involved in opportunistic infections of P. aeruginosa , and mouse macrophage galactose-type lectin (mMGL-2), a protein expressed on antigen presenting cells that may be useful as a vaccine targeting receptor. An advantage of the approach is that structural information about the lectin/receptor is not required to obtain a multivalent inhibitor/probe.

Aptamer-based carbohydrate recognition.

Carbohydrates have been revealed to play fundamental roles in diverse biological phenomena, such as being recognition sites and biomarkers. Recently, ample nucleic acid aptamers guided for carbohydrate recognition have already been isolated and characterized through SELEX methodology. This review would like to present and discuss these aptamers toward recognizing various carbohydrate targets: monosaccharides, oligosaccharides, polysaccharides, aminoglycoside antibiotics and glycans from glycoproteins. High affinity carbohydrate aptamers that we reviewed herein might shed light on the development of a tool to augment drug discovery creations.

Mechanism of the inhibitory action of chestnut astringent skin extract on carbohydrate absorption.

Chestnut astringent skin (CAS) extract inhibited pancreatic alpha-amylase and intestinal alpha-glucosidase in a concentration-dependent manner with the 50% inhibition concentration (IC50) for amylase, maltase and sucrase being 7.5, 650 and 390 microg/mL, respectively. We have investigated the effect of CAS extract on carbohydrate absorption in normal rats. Oral administration of CAS extract to rats fed cornstarch (2 g/kg body weight) significantly suppressed the increase of blood glucose levels and the area under the curve (AUC). Administration of CAS extract to rats fed maltose or sucrose delayed the increase of blood glucose level and slightly suppressed AUC, but not significantly. Administration of CAS extract to rats fed glucose did not affect the increase in blood glucose level or AUC. Similar results were observed with type-2 diabetic model rats (GK/jcl). To test the effect of CAS extract on diabetes, type 2 diabetic model mice (db/db mice) were fed a standard laboratory diet containing 1 or 2% CAS extract. CAS extract prevented increases in body weight and fasting blood glucose concentration. These data suggest that CAS extract has an anti-diabetic function in type 2 diabetic mice that mainly functions through inhibition of alpha-amylase.

Pharmacokinetics of creatine.

Research has demonstrated that creatine supplementation has some therapeutic benefit with respect to muscle function and more recently neurological function. Despite the growing body of literature on the pharmacologic effect of creatine, very little is known about the disposition of creatine after supraphysiologic doses. The movement of creatine throughout the body is governed by transport processes which impact the absorption of creatine from the intestine, clearance of creatine from the kidney, and access of creatine to target tissues. With repeated doses of creatine, it appears that the clearance of creatine decreases mainly due to the saturation of skeletal muscle stores. Insulin and insulin-stimulating foods appear to enhance muscle uptake of creatine but at the same time, high carbohydrate meals may slow the absorption of creatine from the intestine. Little is known about creatine disposition in special populations including the elderly and patients with neuromuscular disease. Knowledge of creatine disposition in these clinically relevant populations can help remove some of the guess work of dose selection during clinical trials.

RNA aptamers directed against oligosaccharides.

Nucleic acid molecules are designed to interact predominantly with proteins or complementary nucleic acids. Interaction of nucleic acids with carbohydrates, abundant constituents of glycoproteins and glycolipids, are not common in cells. Biomedical applications of nucleic acids targeted against oligosaccharides, which are involved in the function of receptors, immune answer, host interaction with invading infectious agents, and cancer metastasis, are feasible. In vitro selection of nucleic acids interacting with oligoand polysaccharides is a promising strategy to identify potential inhibitors of biochemical recognition processes in which carbohydrates are involved. Several RNA and DNA aptamers directed against carbohydrates have already been isolated and characterized. The results are summarized in this article, and an attempt is made to draw initial conclusions concerning the perspectives of the outlined approach.

New glycomimetics: anomeric sulfonates, sulfenamides, and sulfonamides.

The synthesis of a variety of new 1-thio-D-glucopyranose derivatives oxidized at the sulfur atom is described, including seven 1-C-sulfonic acids, three sulfonate esters, three sulfinate esters, an S,S'-diglycosyl thiolsulfonate and thiolsulfinate, four S-glycosyl sulfenamides, an S-glycosyl sulfinamide, and two S-glycosyl sulfonamides. These compounds possess unusual anomeric functionality that might be resistant or even inhibitory to normal enzymatic carbohydrate processing, and therefore, they may be of future use in studies of enzyme inhibition, structure, mechanism, and function.

The design, synthesis and evaluation of high affinity macrocyclic carbohydrate inhibitors.

Carbohydrate-protein interactions have been investigated for a model system of a monoclonal antibody, SYA/J6, which binds a trisaccharide epitope of the O-polysaccharide of the Shigella flexneri variant Y lipopolysaccharide. The thermodynamics of binding for the methyl glycoside of the native trisaccharide epitope, Rha-Rha-GlcNAc () to SYA/J6 over a range of temperatures exhibits strong, linear enthalpy-entropy compensation and a negative heat capacity change (DeltaC(p)=-152 cal mol(-1) degree(-1)). At 293 K the free energy of association is the sum of favourable enthalpy and entropy contributions (DeltaH=-3.9 kcal mol(-1) and -TDeltaS=-2.9 kcal mol(-1)). Crystal structures for SYA/J6 Fab detailed the position of the native trisaccharide epitope, Rha-Rha-GlcNAc, and facilitated a strategy to design a tighter binding, low molecular weight ligand. This involved pre-organization of the native trisaccharide in its bound conformation by addition of intramolecular constraints (a beta-alanyl or glycinyl tether). ELISA measurements indicated that the glycinyl tethered trisaccharide was not an optimal candidate for further analysis, while microcalorimetry provided data showing that the beta-alanyl tethered trisaccharide displayed a 15-fold increase in affinity for SYA/J6. Tethering resulted in a favourable entropic contribution to binding, relative to the native trisaccharide (-TDeltaDeltaS=-1.2 kcal mol(-1)). Potential energy and dynamics calculations using the AMBER Plus force fields indicated that trisaccharide adopted a rigid conformation similar to that of the bound conformation of the native trisaccharide epitope. While this strategy resulted in modest free energy gains by minimizing losses due to conformational entropy, thermodynamic data are consistent with significant contributions from solvent reorganization.

Beta-galactosidase and alpha-mannosidase inhibit formation of multicellular nodules in breast cancer cell cultures.

In response to an estrogen, confluent monolayers of MCF-7 cell cultures develop multi-cellular nodules, termed foci. Post-confluent development of foci occurs with physiologic levels of 17beta-estradiol and are inhibited by various anti-estrogens acting through either the estrogen or aryl hydrocarbon receptors. In the present paper we report that disruption of the terminal sugars on membrane receptors results in inhibition of foci. Treatment with 0.013-0.05 units/ml of beta-galactosidase completely inhibited the development of foci while leaving the monolayer of cells intact. Trials with alpha-mannosidase resulted in a similar but less potent inhibition of foci. Lectin-fluorescent conjugates, RCA (Ricinus communis agglutinin), and ConA (Canavalia ensiformis agglutinin) were used to identify membrane surface carbohydrates on MCF-7 cells. Binding of the RCA-fluorescent conjugate was inhibited by co-treatment with galactose or lactose. Binding of ConA-fluorescent conjugate was significantly inhibited by mannose and n-acetyl-glucosamine. This is the first report of inhibition of foci development in MCF-7 cell cultures by disruption of surface carbohydrates on membrane receptors.

Structural basis for the inhibition of mammalian and insect alpha-amylases by plant protein inhibitors.

Alpha-amylases are ubiquitous proteins which play an important role in the carbohydrate metabolism of microorganisms, animals and plants. Living organisms use protein inhibitors as a major tool to regulate the glycolytic activity of alpha-amylases. Most of the inhibitors for which three-dimensional (3-D) structures are available are directed against mammalian and insect alpha-amylases, interacting with the active sites in a substrate-like manner. In this review, we discuss the detailed inhibitory mechanism of these enzymes in light of the recent determination of the 3-D structures of pig pancreatic, human pancreatic, and yellow mealworm alpha-amylases in complex with plant protein inhibitors. In most cases, the mechanism of inhibition occurs through the direct blockage of the active center at several subsites of the enzyme. Inhibitors exhibiting "dual" activity against mammalian and insect alpha-amylases establish contacts of the same type in alternative ways.

Role of carbohydrates in repair of human respiratory epithelium using an in vitro model.

BACKGROUND: The epithelial layer in the conducting airway provides a primary protective barrier. Repair of this barrier normally occurs rapidly after damage, but is compromised in diseases such as asthma. OBJECTIVE: We have developed a human in vitro model system to test our hypothesis that cell surface glycoconjugate-based interactions are required for the normal repair of damaged epithelium. METHODS: Lectins having narrow carbohydrate specificities were used to identify and block specific carbohydrate moieties on human airway-derived epithelial cells in culture. RESULTS: The lectin wheat germ agglutinin bound to N-acetyl glucosamine and inhibited the repair of epithelial damage while having little effect on cell viability. In contrast, other N-acetyl glucosamine binding lectins had no effect even when bound to the cell surface. The involvement of glycoconjugates was confirmed by pre-incubating the lectin with its specific sugar, preventing the inhibition of repair. CONCLUSION: These results indicate that lectin-binding sites are involved in epithelial repair and may be important in the repetitive cycles of injury and repair seen in asthma. This model system provides an insight into the role of glycoconjugates and will help to determine the function of specific carbohydrate groups in epithelial repair. These may present a target for therapeutic intervention in respiratory and other diseases.

A homogeneous cell-based assay to identify N-linked carbohydrate processing inhibitors.

Malignant transformation is accompanied by altered cell surface glycosylation. N-Linked oligosaccharides carrying beta1-6GlcNAc branches are associated with tumor invasion and metastasis. Therefore, compounds that can enter cells and block biosynthesis of beta1-6GlcNAc-branched glycans without overt cytotoxicity are potential anticancer agents. We have developed a homogeneous cell-based assay for detection of such compounds. The method enables identification of agents that block beta1-6GlcNAc-branched glycan expression after incubation for 16-20 h with MDAY-D2 tumor cells, thereby protecting the cells from the subsequent addition of leukoagglutinin, a cytotoxic plant lectin. We observed that MDAY-D2 cell number is directly proportional to the level of endogenous alkaline phosphatase activity measured spectrophotometrically in cultures after the addition of substrate. The alkaline phosphatase assay was capable of detecting as few as 1,500 cells. The assay was readily adapted for high-throughput screening as reagent costs are low and no cell harvesting and washing steps are required. Under high-throughput operating conditions, the coefficient of variation for controls was found to be 4.2%. The results suggest that measurement of alkaline phosphatase in this cell assay format may be adapted for wider applications in high-throughput screenings for compounds that relieve cells from other growth inhibitors.

N-linked carbohydrate on human leukocyte antigen-C and recognition by natural killer cell inhibitory receptors.

The possible role of carbohydrate in the interaction of HLA-C with a human inhibitory natural Killer cell Immunoglobulin-like Receptor with two Ig domains, KIR2DL1, was investigated. Transfectants of 721.221 (a class I MHC-negative human B cell line) expressing only HLA-Cw4 or -Cw6 or their respective non-glycosylated mutants (N86Q, S88A) were made. The binding of a KIR2DL1-Ig fusion protein to the non-glycosylated mutant HLA-Cw4- or -Cw6-expressing cells was markedly decreased compared to the wild type-expressing cells. The ability to induce an inhibitory signal in the NK tumor line YTS transfected with KIR2DL1 was also impaired in the nonglycosylated mutant expressing cells. Furthermore, in a second functional assay, mutant HLA-Cw4 and -Cw6 molecules had impaired ability to induce signal transduction in BW cells expressing a KIR2DL1-CD3 zeta chain chimeric protein. Thus, the deletion of the N-linked glycosylation signal in HLA-Cw4 and -Cw6 greatly reduced recognition by KIR2DL1. Alternative interpretations of the data are discussed.

Inhibition of salivary amylase by black and green teas and their effects on the intraoral hydrolysis of starch.

Tea decoctions prepared from a number of black and green teas inhibited amylase in human saliva. Black teas gave higher levels of inhibition than green teas, and removal of tea tannins with gelatin led to the loss of inhibitory activity from all decoctions. Streptococcal amylase was similarly inhibited by tea decoctions. Fluoride was without effect on amylase. Since salivary amylase hydrolyzes food starch to low molecular weight fermentable carbohydrates, experiments were carried out to determine whether tea decoctions would interfere with the release of maltose in food particles that became entrapped on the dentition. Subjects consumed salted crackers and rinsed subsequently for 30 s with black or green tea decoctions, or water. Maltose release was reduced by up to about 70% after rinsing with the teas. Black tea decoction was significantly more effective than green tea, in agreement with the in vitro data. The observations supported the hypothesis that tea consumption can be effective in reducing the cariogenic potential of starch-containing foods such as crackers and cakes. Tea may reduce the tendency for these foods to serve as slow-release sources of fermentable carbohydrate.

Identification of a sorbitol permease in human erythrocytes.

Sorbitol, a polyol derived from glucose by the enzyme, aldose reductase, is a common organic solute in many cells. It plays a role in the osmotic regulation of epithelial cells and in the pathology of uncontrolled diabetes. To learn more about sorbitol transport, we measured D-[14C]sorbitol influx in human erythrocytes. Sorbitol influx at 37 degrees C was a linear function of sorbitol concentration over the range of 0.05-100 mM. The activation energy for sorbitol influx was 10.0 kcal/mol, and the Q10 over the range 10-50 degrees C was 1.8, higher than predicted for diffusion through an aqueous pore. Glucose transport inhibitors either had no effect (1 mM phloridzin) or minimally inhibited (approximately 35% inhibition by 10 microM cytochalasin B or 250 microM phloretin) sorbitol influx. Influx was stimulated twofold by 0.5 mM p-chloromercuribenzoic acid, an inhibitor of glucose transport, and this was reversed by 2 mM dithiothreitol. Sorbitol influx was neither Na dependent nor sensitive to changes in cell volume. Glucose, fructose, mannitol, myo-inositol, and gluconate, at four- to fivefold molar excesses over sorbitol, did not inhibit its influx. We conclude that there is a specific sorbitol transport pathway in human erythrocytes similar to the sorbitol permease in renal epithelial cells.