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.

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.

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.

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.

Differential permeability of the proximal and distal rabbit small bowel.

The permeability of the proximal and distal rabbit intestine for two to six carbon polyhydric alcohols was compared. Intestinal segments were mounted in chambers that permitted the measurement of the unidirectional flux across the brush border membrane. For both proximal and distal intestine, the permeability for a series of polyhydric alcohols decreased with increasing size. The proximal intestine was more permeable for four, five, and six carbon polyhydric alcohols than distal intestine. This regional permeability difference can be attributed to variations in the permeability characteristics of the brush border specifically. The uptake of alcohols was nonsaturable and was not inhibited by phlorizine or n-ethylmaleimide. The results are compatible with the concept that the brush border membrane has properties similar to artificial porous membranes and that the equivalent radius of the pores of the proximal intestine exceeds that of the distal gut.

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.

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.

Analysis of carbohydrate properties essential for melanogenesis in tyrosinases of cultured malignant melanoma cells by differential carbohydrate processing inhibition.

In order to clarify the biologic significance of carbohydrate processing in tyrosinases for melanogenesis, we have studied the effect of differential carbohydrate processing inhibitors on the recovery process of interrupted melanization which occurs after exposure of cultured B-16 melanoma cells to the inhibitor of core carbohydrate synthesis, glucosamine (Glc). Treatment of this glycosylation-dependent repigmentation process with the early-stage carbohydrate processing inhibitors deoxynojirimycin (dNM), castanospermine (CS), and monensin (MS) at 0.8 mM, 40 micrograms/ml, and 30 nM, respectively, in the presence of 2 mM theophylline (Tp) almost completely inhibits the reappearance of the pigment 48-72 h after removal of Glc. In contrast, treatment with the later stage carbohydrate processing inhibitor swaisonine (SW) at 40-80 micrograms/ml does not interrupt the repigmentation process. Electrophoretic analysis of tyrosinases in the soluble fractions of these melanoma cells demonstrates that the alteration of soluble tyrosinase isozymes by all the processing inhibitors is associated with a dose-dependent loss of sialic acid-rich T1 tyrosinase and the concomitant appearance or increase of sialic acid-poor tyrosinases. In the large granule fraction, a recovery of membrane-bound tyrosinase (T3) is seen following both MS and SW treatments, whereas dNM treatment results in the substantial loss of T3 tyrosinase. At the electron microscopic level, a translocation of tyrosinase from GERL and coated vesicles to many unmelanized vacuolar premelanosomes occurs in MS-treated cells in contrast to its predominant distribution in the GERL-coated vesicle system of dNM-treated cells, which contain many unmelanized premelanosomes. The present evidence for differential effects on intracellular tyrosinase transfer and melanization by different stages of carbohydrate processing inhibition suggests that asparagine-linked oligosaccharides, relating to the first mannose-trimming stages, determine the function of tyrosinase transfer as well as melanization through a specific intracellular recognition process in pigment cells.

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.

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.

Hemagglutinating activity of Dolichos lablab seed protein.

The hemagglutinating activity of a crude extract and partially purified protein fractions from seeds of Dolichos lablab grown in the state of Bahia, Brazil, has been examined. The crude extract agglutinates rabbit erythrocytes non-specifically with respect to blood groups. The hemagglutination of rabbit erythrocytes induced by the crude extract of D. lablab was inhibited most effectively by N-acetyl-D-glucosamine (3 mM) and alpha-methyl-D-mannoside (3 mM) followed by D-glucosamine (6 mM), D-mannose (6 mM) and D-glucose (12 mM). The spectrum of sugar inhibition of the hemagglutinating activity of the Brazilian seeds combines the characteristics of seeds coming from Turkey and India as indicated by the preferential inhibitory effect exerted by N-acetyl-D-glucosamine and the inhibition by D-mannose and D-glucose. These data suggest that the Brazilian seeds may belong to a different variety of Dolichos lablab.

[Study of the inhibitory action of phenothiazine on carbohydrate metabolism in Mecistocirrus digitatus (Nematoda: Rhabditida)]. / Izuchenie ingibitornogo deistviia fenotiazina na obmen uglevodov u Mecistocirrus digitatus (Nematoda: Rhabditida)

The presence of phenothiazine in incubation medium of M. digitatus exerted the following effects: the increase of glycogene splitting and lactic acid excretion in aerobic conditions; the increase of acetic acid excretion and the decrease of alpha-methylbutyric acid excretion; glycolysis process inhibition in homogenates and supernatants during incubation with glucose. However, at phenothiazine incubation with FDF glycolysis intensity was unaffected.

[Effect of the time of ethrel use on the sugar levels in onions]. / Wplyw terminu stosowania ethrelu na poziom cukrów w cebuli.

Two groups of onion plants were treated with Ethrel on two different dates; onions were then stored, and the changes in their sugar contents were compared. Two onion cultivars: Czerniakowska and Wolska, were investigated in simple sugar contents between treated onions (on the I and II date) and untreated controls. Mean contents of simple sugars were lower in onions treated on the I date, as compared with the II date. Ethrel treatment (on the I and II date) caused a decrease in oligosugar contents; this drop amounted, respectively, for the Czerniakowska cultivar to 14.3 and 15.4% and for the Wolska cultivar to 15.8 and 18.8%. Ethrel treatment, as compared with control, significantly decreased total sugar contents; this decrease was for the Czerniakowska cultivar 4.8-7.6%, and for the Wolska cultivar 7.2-7.6%. The date of Ethrel treatment exerted no univocal effect on the decrease in oligosugars and total sugars.

[The carbohydrate-resistant mechanism of the action of caries-prophylactic agents on the oral fluid in children]. / Uglevodrezistentnyi mekhanizm deistviia kariesprofilakticheskikh sredstv na sostoianie rotovoi zhidkosti u detei.

Examinations of 176 children administered caries-preventing drugs for 2 years have shown that oral irrigation with 0.2% sodium fluoride solution, oral intake of fluorine in a dose of 1 mg, or of potassium orotate, or of calcium glucerophosphate improved the oral fluid resistance to carbohydrate action. Glycolytic enzyme activity of the oral fluid in these children was not augmenting during carbohydrate load, whereas in children not administered such preventive courses oral intake of 10% glucose solution resulted in essential elevation of salivary aldolase and lactate dehydrogenase activities, i.e. intensification of glycolytic processes, this leading to the development of acid potential in the oral cavity and, consequently, to a higher risk of caries development.

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.

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.