α-Oligoglucosylation of a sugar moiety enhances the bioavailability of quercetin glucosides in humans

Dietary intake of quercetin is suggested to be potentially beneficial for the prevention of various diseases. We examined the effect of α-oligoglucosylation of the sugar moiety of quercetin monoglucoside on its bioavailability in humans. Enzymatically modified isoquercitrin (EMIQ) was prepared by enzymatic deglycosylation and the subsequent of α-oligoglucosylation of quercetin 3-O-β-rutinode (rutin). The plasma level of quercetin metabolites was instantly increased by oral intake of EMIQ and its absorption efficiency was significantly higher than that of isoquercitrin (quercetin 3-O-β-glucoside; Q3G), and rutin. The profile of plasma quercetin metabolites after EMIQ consumption did not differ from that after Q3G consumption. The apparent log P of EMIQ indicated that EMIQ is more hydrophilic than Q3G but less than quercetin 3,4′-O-β-diglucoside. These data indicated that enzymatic α-oligoglucosylation to the sugar moiety is effective for enhancing the bioavailability of quercetin glucosides in humans.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Structural studies of the acidic oligosaccharides

Among the four acidic oligosaccharide fractions obtained by paper electrophoresis of the hydrazinolysate of the plasma membrane glycoproteins of rat erythrocytes, one was further separated into two by prolonged paper electrophoresis using 120-cm paper. Three fractions were mixtures of monosialyl oligosaccharides and two of disialyl oligosaccharides. After desialylation, their neutral portions were fractionated by Bio-Gel P-4 column chromatography and by affinity chromatography using a Con A-Sepharose column. Structural studies of the neutral oligosaccharides, thus obtained, indicated that at least 26 different complex-type oligosaccharides are present as a neutral portion of the acid oligosaccharides. Structurally they can be classified into bi-, tri-, and tetraantennary oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc_OT as their common cores. Galβ1 → 3Galβ1 → 4GlcNAc, Siaα2 → 3Galβ1 → 4GlcNAc, Siaα2 → 6Galβ1 → 4GlcNAc, and a series of Siaα2 → (Galβ1 → 4GlcNAcβ1 → 3)_n · Galβ1 → 4GlcNAc were found as their outer chains. Their structures together with the structures of neutral oligosaccharides reported in the preceding paper indicated that the outer chain moieties of the asparagine-linked sugar chains of rat erythrocyte membrane glycoproteins are formed not by random concerted action of glycosyl transferases in Golgi membrane but by the mechanism in which the formation of one outer chain will regulate the elongation of others.     

Photoperiodic control of sugar release during the floral transition: What is the role of sugars in the florigenic signal?

Florigen is a mobile signal released by the leaves that reaching the shoot apical meristem (SAM), changes its developmental program from vegetative to reproductive. The protein FLOWERING LOCUS T (FT) constitutes an important element of the florigen, but other components such as sugars, have been also proposed to be part of this signal.^1-5 We have studied the accumulation and composition of starch during the floral transition in Arabidopsis thaliana in order to understand the role of carbon mobilization in this process. In A. thaliana and Antirrhinum majus the gene coding for the Granule-Bound Starch Synthase (GBSS) is regulated by the circadian clock^6,7 while in the green alga Chlamydomonas reinhardtii the homolog gene CrGBSS is controlled by photoperiod and circadian signals.^8,9 In a recent paper¹⁰ we described the role of the central photoperiodic factor CONSTANS (CO) in the regulation of GBSS expression in Arabidopsis. This regulation is in the basis of the change in the balance between starch and free sugars observed during the floral transition. We propose that this regulation may contribute to the florigenic signal and to the increase in sugar transport required during the flowering process.     

SPR and ITC determination of the kinetics and the thermodynamics of bivalent versus monovalent sugar ligand–lectin interactions

A kinetic study of the interaction of bivalent and monovalent sugar ligands with a lectin was undertaken with the aid of surface plasmon resonance (SPR) method. The study involved a series of bivalent α-d-mannopyranoside containing sugar ligands, with systematic variation in the distance between the sugar ligands. The detailed kinetic studies showed that bivalent ligands underwent a faster association (k _on) and a slower dissociation (k _off) of the ligand–lectin complexes, in comparison to the monovalent ligand–lectin complexes. The kinetic constants were complemented further by assessing the thermodynamic parameters with the aid of isothermal titration calorimetry (ITC). The initiation of cross-linking of ligand–lectin interactions emerge from the early stages of the complexation. The dynamic light scattering (DLS) and the transmission electron microscopy (TEM) techniques allowed judging the sizes and morphologies of the complex in the solution and solid states, respectively.     

Do additional sugar sources affect the degree of attendance of Dysmicoccus brevipes by the fire ant Solenopsis geminata?

Mutualistic interactions between ants and Hemiptera are mediated to a large extent by the amount and quality of sugar‐rich honeydew produced. Throughout the neotropics, the predaceous fire ant Solenopsis geminata (Fabricius) (Hymenoptera: Formicidae) is found in association with colonies of the pineapple mealybug, Dysmicoccus brevipes (Cockerell) (Hemiptera: Pseudococcidae), which they actively tend and protect from attack by natural enemies. In this study, we evaluate the effects of access to a sucrose solution on the mutualistic association between S. geminata and D. brevipes. Ten colonies of either species were established, with D. brevipes maintained on pumpkin, Cucurbita maxima Duchesne (Cucurbitaceae), in screen cages. Five of the S. geminata colonies were permitted access to vials with 20% sucrose solution and a pumpkin with 20 adult mealybugs. The remaining ant colonies were allowed access to mealybug‐infested pumpkins. Ant colonies with access to the sucrose solution attended mealybugs significantly less than those without additional sugar sources. Mealybug survival rates were similar under both treatments. Total body sugars and fructose were nearly twice as high in ants with access to honeydew and sucrose vs. those with access to honeydew and water. Fructose accumulated on the pumpkins over time in both treatments, suggesting that honeydew was not fully exploited by the ants. In conclusion, D. brevipes enjoy lower degrees of ant attendance when S. geminata have alternative sources of carbohydrates. We further discuss the significance of these findings for the conservation of predaceous ants and mealybug biological control.     

Structure identification of the complex-type,asparagine-linked sugar chains of β-d-galactosyl-transferase purified from human milk

The asparagine-linked sugar chains of human milk galactosyltansferase were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted into radioactive oligosaccharides by sodium borotritiate reduction after N-acetylation, and fractionated by paper electrophoresis and by Bio-Gel P-4 column chromatography after sialidase treatment. Structural studies of each oligosaccharides by sequential exoglycosidase digestion and methylation analysis indicated that the galactosyltransferase contains bi, tri-, and probably tetra-antennary, complex-type oligosaccharides having α-d-Manp-(1→3)-[α-d-Manp-(1→6)]-β-d-Manp-(1→4)-β-d-GlcpNAc-(1→4)-α-d-[Fucp-(1→6)]-d- GlcNAc as their common core. Variation is produced by the different locations and numbers of the five different outer chains: β-d-Galp-(1→4)-d-GlcNAc, α-l-Fucp-(1→3)-[β-d-Galp-(1→4)]-d-GlcNAc, α-NeuAc-(2→6)-β-d-Galp-(1→4)-d-GlcNAc, α-l-Fucp-(1→3)-[β-d-Galp-(1→4)]-β-d-GlcpNAc-(1→3)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d- GlcNAc, and α-NeuAc-(2→6)-β-d-Galp-(1→4)-β-d-GlcpNAc-(1→3)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)-β-d-GlcNAc.     

Gas chromatographic–mass spectrometric analysis of urinary sugar and sugar alcohols during pregnancy

A refined and simplified method has been developed for the simultaneous analysis of urinary sugar and sugar alcohols after urease treatment by using capillary gas chromatography–mass spectrometry (GC–MS). Since carbohydrate metabolism during pregnancy is considered to be diabetogenic, our interest has been concentrated on understanding the mechanism of the metabolic deviation by assessing the glucose excursion and glucose fluxes. The present study suggests that changes of the levels of glucose, sorbitol, fructose, myo-inositol, and 1,5-anhydro-D-glucitol (1,5-AG) may reflect a mild alteration in carbohydrate metabolism that goes undetected by conventional diabetic indicators.     

Immobilized α-d-galactosidase in the sugar beet industry

Mycelia containing α-galactosidase have been used commercially for about ten years to hydrolyse raffinose in beet sugar molasses to aid in the production of sugar. The development of α-galactosidase-producing strains of three genera of fungi, the production of the enzyme and its use in beet sugar manufacture are reviewed. The treatment of mycelial pellets containing the α-galactosidase with glutaraldehyde and the consequent stabilization of the activity are described.     

The “epimerring” highlights the potential of carbohydrate epimerases for rare sugar production

Rare sugars can find applications in various industrial sectors and, therefore, hold significant economic value. Due to their low natural abundance, efficient production processes are needed to enable their commercial exploitation. About a decade ago, the available biosynthetic routes were summarized in the so-called “Izumoring”, which mainly comprised reactions catalysed by keto-aldol isomerases and oxidoreductases. Although just a single epimerase specificity (acting on the 3-position of ketoses) was included, these enzymes hold the potential to truly revolutionize the field as they offer shortcuts in conversion processes. For example, C2-epimerases could replace double isomerization reactions, whereas C4/5-epimerases could form a new bridge between d- and l-sugars as alternative to the current two-step oxidoreduction reaction. Here, we present the “Epimerring” to highlight the potential of new epimerases that can still be discovered and/or engineered, which may open doors to new and improved synthesis routes for rare sugars. Several efforts and options in the search of such biocatalysts are shortly summarized.     

The effect of the sugar metabolism on Leishmania infantum promastigotes inside the gut of Lutzomyia longipalpis: A sweet relationship?

It is well-known that Leishmania parasites can alter the behavior of the sand fly vector in order to increase their transmission potential. However, little is known about the contribution of the infecting host’s blood composition on subsequent sand fly infection and survival. This study focused on the host’s glucose metabolism and the insulin/insulin-like growth factor 1 (IGF-1) pathway as both metabolic processes are known to impact vector-parasite interactions of other protozoa and insect species. The focus of this study was inspired by the observation that the glycemic levels in the blood of infected Syrian golden hamsters inversely correlated to splenic and hepatic parasite burdens. To evaluate the biological impact of these findings on further transmission, Lutzomyia longipalpis sand flies were infected with blood that was artificially supplemented with different physiological concentrations of several monosaccharides, insulin or IGF-1. Normoglycemic levels resulted in transiently higher parasite loads and faster appearance of metacyclics, whereas higher carbohydrate and insulin/IGF-1 levels favored sand fly survival. Although the recorded effects were modest or transient of nature, these observations support the concept that the host blood biochemistry may affect Leishmania transmission and sand fly longevity.     

Identification of the first fluorescent α-amidoboronic acids that change fluorescent properties upon sugar binding

The first amidoboronic acids were identified that show significant fluorescent property changes upon binding with various carbohydrates.     

Glycoproteins: what are the sugar chains for?

For many glycoproteins, the carbohydrate groups confer important physical properties such as conformational stability, protease resistance, charge and water-binding capacity. Equally important, however, are the roles of carbohydrate groups in biological recognition, where sequence diversity provides signals for protein targeting and cell-cell interactions.     

Properties of membrane-bound α-glucosidases: Possible sugar receptor protein of the blowfly, Phormia regina

Previously reported PII-type α-glucosidase located in the precipitate of the labellar homogenate of the blowfly Phormia regina was solubilized by sodium deoxycholate (DOC) and further separated into three isozymes with different molecular weight: PII-M (mol. wt 9 × 10⁴). PII-D (mol. wt 2 × 10⁵) and PII-T (mol. wt 8 × 10⁵) by molecular sieve chromatography on Biogel P-300 or Ultragel AcA-34. These three isozymes had almost the same K_m's and relative values of V_m's for several substrates, suggesting that they had the same common active site.PII-D and PII-T are more strongly embedded in the membrane than PII-M, because the proportion of PII-D and PII-T was much increased when the remaining glucosidase in the precipitate after the first solubilization was reextracted by DOC. A large peak of α-glucosidase isozyme P-IV which preferentially hydrolyze sucrose eluted just after P-II (soluble P-II) when the supernatant fraction of the labellar homogenate was chromatographed on DEAE-Sephadex A-50. P-IV was scarcely present in the precipitate fraction.Soluble P-II had the same mol. wt as PII-M and had similar properties to PII-M except for the ratio of V_m's.A large proportion of PII-D was contained in the well washed labellar integuments, a preparation rich in labellar chemosensilla. It suggests that most of the insoluble α-glucosidase contained in the dendrite in labellar chemosensilla is PII-D. PII-D (and PII-T) are possible sites of the pyranose receptor molecule because their properties and localization agree well with those of the receptor.     

TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor

Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [¹⁵N-¹H]- and [¹³C-¹H]-methyl-TROSY NMR spectra with a 52?kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48?h at a temperature of 25?°C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan ¹⁵N backbone positions and also resolved signals for ¹⁵N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [¹³C-¹H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.     

Is the onset of senescence in leaf cells of intact plants due to low or high sugar levels?

This review examines the hypotheses that developmental programmed cell death in leaves is mediated (i) by sugar starvation in the leaf cells or (ii) by sugar accumulation in these cells. Experimental evidence for both hypotheses is critically discussed and found to be lacking. For example, some papers show that sugars prevent senescence of cut leaves placed in darkness, and prevent low sugar levels in the leaves. In these tests, the sugars seem to replace photosynthesis, hence the results have little relevance to leaf senescence in intact plants in the light. Low nitrogen nutrition and high light results in earlier senescence than the low nitrogen treatment alone. This is accompanied by high sugar levels in the leaves. The results have led to the idea that accumulation of sugars is the cause of the additional effect, or more generally, that sugar accumulation is always the direct cause of leaf senescence. Results from over-expressing, or knocking out, hexokinase genes tend to support the high sugar hypothesis, but pleiotropic effects confound this conclusion. In addition, several experiments show the effects of treatments on senescence without the increase in leaf sugar levels. Nonetheless, sugar levels are usually measured in whole leaves. Such an overall level does not reflect the differences in the onset of senescence between tissues and cells, and can therefore not be used as an argument for or against either of the two hypotheses. It is argued that future work should determine the time line of the concentrations of various sugars in various cells and cellular compartments, in relation to senescence processes in the same cells. Taken together, the data are not decisive. It is possible that neither of the two hypotheses is correct.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Fractionation of oligosaccharides liberated by hydrazinolysis and structural studies of the neutral oligosaccharides

The asparagine-linked sugar chains of the plasma membrane glycoproteins of rat erythrocytes were released as oligosaccharides by hydrazinolysis and labeled by NaB³H₄ reduction. The radioactive oligosaccharides were separated into a neutral and at least four acidic fractions by paper electrophoresis. The neutral oligosaccharide fraction was separated into at least 11 peaks upon Bio-Gel P-4 column chromatography. Structural studies of them by sequential exoglycosidase digestion in combination with methylation analysis revealed that they were a mixture of three high mannose-type oligosaccharides and at least 11 complex type oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their cores and Galβ1 → 4GlcNAc, Galβ1 → 3Galβ1 → 4GlcNAc, and various lengths of Galβ1 → 4GlcNAc repeating chains in their outer chain moieties. Most of the complex-type Oligosaccharides were biantennary, and the tri- and tetraantennary Oligosaccharides contain only the Galβ1 → 3Galβ1 → 4GlcNAc group in their outer chain moieties.