Linked Glucose Polymer Research Articles

Antisense Oligonucleotides Selectively Enter Human-Derived Antibiotic-Resistant Bacteria through Bacterial-Specific ATP-Binding Cassette Sugar Transporter.

Current vehicles used to deliver antisense oligonucleotides (ASOs) cannot distinguish between bacterial and mammalian cells, greatly hindering the preclinical or clinical treatment of bacterial infections, especially those caused by antibiotic-resistant bacteria. Herein, bacteria-specific ATP-binding cassette (ABC) sugar transporters are leveraged to selectively internalize ASOs by hitchhiking them on α (1-4)-glucosidically linked glucose polymers. Compared with their cell-penetrating peptide counterparts, which are non-specifically engulfed by mammalian and bacterial cells, the presented therapeutics consisting of glucose polymer and antisense peptide nucleic-acid-modified nanoparticles are selectively internalized into the human-derived multidrug-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus, and they display a much higher uptake rate (i.e., 51.6%). The developed strategy allows specific and efficient killing of nearly 100% of the antibiotic-resistant bacteria. Its significant curative efficacy against bacterial keratitis and endophthalmitis is also shown. This strategy will expand the focus of antisense technology to include bacterial cells other than mammalian cells.

Automated Assembly of Starch and Glycogen Polysaccharides

Polysaccharides are Nature’s most abundant biomaterials essential for plant cell wall construction and energy storage. Seemingly minor structural differences result in entirely different functions: cellulose, a β (1–4) linked glucose polymer, forms fibrils that can support large trees, while amylose, an α (1–4) linked glucose polymer forms soft hollow fibers used for energy storage. A detailed understanding of polysaccharide structures requires pure materials that cannot be isolated from natural sources. Automated Glycan Assembly provides quick access to trans-linked glycans analogues of cellulose, but the stereoselective installation of multiple cis-glycosidic linkages present in amylose has not been possible to date. Here, we identify thioglycoside building blocks with different protecting group patterns that, in concert with temperature and solvent control, achieve excellent stereoselectivity during the synthesis of linear and branched α-glucan polymers with up to 20 cis-glycosidic linkages. The molecules prepared with the new method will serve as probes to understand the biosynthesis and the structure of α-glucans.

Insights into extracellular dextran formation by Liquorilactobacillus nagelii TMW 1.1827 using secretomes obtained in the presence or absence of sucrose

Dextrans are α-(1,6)-linked glucose polymers, which are exclusively produced by lactic acid bacteria from sucrose via extracellular dextransucrases. Previous studies suggested that the environmental pH and the presence of sucrose can impact the release and activity of these enzymes. To get deeper insight into this phenomenon, the dextransucrase expressed by water kefir borne Liquorilactobacillus (L.) nagelii TMW 1.1827 (formerly Lactobacillus nagelii) was recovered in supernatants of buffered cell suspensions that had been incubated with or without sucrose and at different pH. The obtained secretomes were used to time-dependently produce and recover dextrans, whose molecular and macromolecular structures were determined by methylation analysis and AF4-MALS-UV measurements, respectively. The initial pH of the buffered cell suspensions had solely a minor influence on the released dextransucrase activity. When sucrose was present during incubation, the secretomes contained significantly higher dextransucrase activities, although the amounts of totally released proteins obtained with or without sucrose were comparable. However, the dextransucrase appeared to be released in lower amounts into the environment if sucrose was not present. The amount of isolable dextran increased up to 24 h of production, although the total sucrose was consumed within the first 10 min of incubation. Furthermore, the sucrose isomer leucrose had been formed after 10 min, while its concentrations decreased over time and the portions of longer isomaltooligosaccharides (IMOs) increased. This indicated that leucrose can be used by L. nagelii TMW 1.1827 to produce more elongated and branched dextran molecules from presynthesized IMOs, while disproportionation reactions on short IMOs may appear additionally. This leads to increasing amounts of high molecular weight dextran in a state of sucrose depletion. These findings reveal new insights into the pH- and sucrose-dependent kinetics of extracellular dextran formation and may be useful for optimization of fermentative and enzymatic dextran production processes.

Identification and comparison of two closely related dextransucrases released by water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827.

Dextransucrases are extracellular enzymes, which are exclusively expressed by lactic acid bacteria (LAB) and produce α-1→6 linked glucose polymers from sucrose. In this study, two dextransucrases derived from water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827 were identified and comparatively investigated. Differences between both proteins mainly arise from an additional C-terminal glucan-binding domain and the presence of a signal motif in the L. nagelii TMW 1.1827 dextransucrase. L. hordei TMW 1.1822 released the enzyme only in the presence of its substrate sucrose in contrast to L. nagelii TMW 1.1827, while both strains functionally expressed the dextransucrases independently of sucrose. Both enzymes could be recovered as crude protein extracts in culture supernatants, as they are not covalently bound to the cell surface. This enabled the formation of dextrans at equal reaction conditions as well as their subsequent structural analysis in terms of molecular structure and molecular weight. The volumetric transglycosylation and hydrolysis activities were distinctly different for both enzymes, which produced O3-branched dextrans with a comparable degree of branching. Moreover, identical oligosaccharides were obtained for both dextrans upon endo-dextranase digestion, while some differences in the polysaccharide fine structures could be identified from the varying portions of certain oligosaccharides. Dextrans synthesized by the dextransucrase released by L. nagelii exhibited an averaged molecular weight (Mw) of 7.9×107 Da, while those produced by the dextransucrase released by L. hordei exhibited an Mw of 6.1×107 Da. Moreover, glycosylation of glucansucrases by LAB was identified for the first time for the released dextransucrase of L. nagelii TMW 1.1827. Our study therefore reveals new molecular insights into how dextransucrases released by water kefir borne L. hordei TMW 1.1822 and L. nagelii TMW 1.1827 contribute to the complex formation of the traditional beverage water kefir.

Structural Characterization, Technological Functionality, and Physiological Aspects of Fungal β-D-glucans: A Review

ABSTRACTβ-D-glucans are a (1→3)-linked glucose polymer with (1→6)-linked side chains and a major component of fungal cell walls. They exhibit structural integrity to the fungal cell wall. In addition, β-glucans are widely used as food adjuvant in food and pharmaceutical industries because of their physico-chemical properties. Several studies have focused on different isolation processes of (1→3) (1→6)-β-glucan that could affect the physico-chemical and functional properties of β-glucan such as chemical composition, solubility, viscosity, hydration properties, and oil binding capacity. Immunological activity is one of the most important properties of β-glucans. Thus, they are effective in inhibiting growth of cancer cells and metastasis and preventing bacterial infection. In humans, β-glucans reduce blood cholesterol, improve glucose absorption by body cells, and so help wound healing. This review described the prebiotic potentiality of fungal β-D-glucans with the objective to detail the methodologies applied for their extraction, their structure and techno-functional properties, and finally their biological effects.

Kinetics of dextran‐independent α‐(1→3)‐glucan synthesis by Streptococcus sobrinus glucosyltransferase I

Glucosyltransferase (GTF)-I from cariogenic Streptococcus sobrinus elongates the α-(1→3)-linked glucose polymer branches on the primer dextran bound to the C-terminal glucan-binding domain. We investigated the GTF-I-catalyzed glucan synthesis reaction in the absence of the primer dextran. The time course of saccharide production during dextran-independent glucan synthesis from sucrose was analyzed. Fructose and glucose were first produced by the sucrose hydrolysis. Leucrose was subsequently produced, followed by insoluble glucan [α-(1→3)-linked glucose polymers] after a lag phase. High levels of intermediate nigerooligosaccharide series accumulation were characteristically not observed during the lag phase. The results from the enzymatic activity of the acceptor reaction for the nigerooligosaccharide with a degree of polymerization of 2-6 and methyl α-D-glucopyranoside as a glucose analog indicate that the activity increased with an increase in the degree of polymerization. The production of insoluble glucan was numerically simulated using the fourth-order Runge-Kutta method with the kinetic parameters estimated from the enzyme assay. The simulated time course provided a profile similar to that of experimental data. These results define the relationship between the kinetic properties of GTF-I and the time course of saccharide production. These results are discussed with respect to a mechanism that underlies efficient glucan synthesis.

Structural and physiological studies on the storage β-polyglucan of haptophyte Pleurochrysis haptonemofera

The storage beta-polyglucan and catabolic enzyme activities of the haptophyte Pleurochrysis haptonemofera were characterized. The storage beta-polyglucan was prepared by the dimethylsulfoxide-extraction method. (13)C- and (1)H-NMR spectroscopy revealed that the polyglucan consists of beta-(1-->3)- and beta-(1-->6)-linked glucose polymers, with a beta-(1-->6)- to beta-(1-->3)-linkage ratio of 1.5. Gel permeation chromatography showed that the molecular weight of the polyglucan is 1.1-8.4 x 10(4) Da, with a peak at 3.4 x 10(4) Da. The degree of polymerization, which was estimated from the amounts of total carbohydrate and reduced ends, was 203, corresponding to 3.3 x 10(4) Da. A method for measurement of the beta-polyglucan in a small amount of liquid culture involving a mixture of beta-glucanases, Westase, was established. The beta-polyglucan was localized in the soluble fraction of cells. The amount of beta-polyglucan per cell increased at the stationary phase under continuous illumination and decreased in the dark, like those of storage alpha-polyglucans, starch of green algae and glycogen of cyanobacteria. The activities of beta-1,3- and beta-1,6-glucanases involved in the degradation of the storage beta-polyglucan were assayed in vitro, both being optimal at pH 5.0. The beta-1,3-glucanase activity, which was detected on active staining after native polyacrylamide gel electrophoresis, was partially purified by ammonium sulfate precipitation and anion exchange chromatography.

Ligation of the lactosylceramide receptor (CDw17) promotes neutrophil migration

Abstract Introduction: B-Glucan, a β-(1–3) linked glucose polymer, enhances neutrophil anti-infective activity by binding to specific receptors including the lectin site of the CR3 integrin (CD11b/CD18) and the glycosphingolipid lactosylceramide. B-glucan promotes neutrophil chemotaxis towards fMLP and C5a. Enhancement of neutrophil migration to fMLP occurs via CR3 through a p38 dependent mechanism. We investigated the mechanism by which β-glucan promotes neutrophil chemotaxis towards C5a. Methods: Migration was assessed by serum agarose overlay technique using chambered slides precoated or not with β-glucan (100μg/ml). Migration of human neutrophils at 37 C for 2 hours was established towards C5a (10 pmol) using PBS as control. Antibody blocking experiments included neutrophil pretreatment with mAbs specific for CR3 or for CDw17. In some experimental groups neutrophils were treated with the p38 MAP kinase inhibitor SB203580 (10mM) prior to assay. The number of chemotaxing cells was quantified by microscopy. Results: The anti-CDw17 mAb but not anti-CR3 mAb obviated the β-glucan promotion. This finding indicates that lactosylceramide is the β-glucan receptor that mediates enhanced chemotaxis towards C5a. Non-specific isotype antibodies did not affect neutrophil migration. Further, p38-inhibited neutrophils did not demonstrate enhanced migration to C5a in the presence of β-glucan. Conclusions: Ligation of lactosylceramide by β-glucan promoted neutrophil chemotaxis towards C5a signaling through a p38 dependent pathway. This is the first demonstration of the involvement of lactosylceramide in neutrophil chemotaxis. This receptor may prove to be a novel therapeutic target in enhancing neutrophil anti-infective activity.

Accumulation of high-molecular-weight amylose in Alzheimer's disease brains.

Although most of the glucose metabolized in the brain is taken up from the blood, glucose derived from glycogen stores is increasingly implicated in both normal brain function and injury repair. An impaired glucose metabolism is one of the features of Alzheimer's disease (AD) entailing a reduction in glucose transporters and the uptake of glucose as well as alterations in the specific activity of glycolytic enzymes. Here we report that AD brains accumulate amylose, the unbranched alpha(1,4)-linked glucose polymer that is resistant to degradation by glycolytic enzymes. Neutral polysaccharides harvested from postmortem brains were purified with hydrazinolysis, ion exchange, and sizing chromatography and subjected to NMR spectroscopy, GC, GC-MS, and methylation analysis. Five percent of the polysaccharides (50 micro g [0.3 micro mol]/g wet weight brain tissue) consisted of amylose with molecular weights exceeding 600,000 Da. There is no evidence for 1,6-branching, indicating that the polymer is not a form of high-molecular-weight glycogen. By GC analysis, the glucose content of the AD brains was almost three times greater than that of the age-matched control brains. A synthesis of amylose in AD brains at the expense of glycogen would compromise glucose metabolism and enhance neural degeneration.

Three pathways for trehalose biosynthesis in mycobacteria.

Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection. To obtain an overview of trehalose metabolism, we analysed data from the Mycobacterium tuberculosis genome project and identified ORFs with homology to genes encoding enzymes from three trehalose biosynthesis pathways previously characterized in other bacteria. Functional assays using mycobacterial extracts and recombinant enzymes derived from these ORFs demonstrated that mycobacteria can produce trehalose from glucose 6-phosphate and UDP-glucose (the OtsA-OtsB pathway) from glycogen-like alpha(1-->4)-linked glucose polymers (the TreY-TreZ pathway) and from maltose (the TreS pathway). Each of the pathways was found to be active in both rapid-growing Mycobacterium smegmatis and slow-growing Mycobacterium bovis BCG. The presence of a disrupted treZ gene in Mycobacterium leprae suggests that this pathway is not functional in this organism. The presence of multiple biosynthetic pathways indicates that trehalose plays an important role in mycobacterial physiology.

Sugar utilization and its control in hyperthermophiles.

Many hyperthermophilic microorganisms show heterotrophic growth on a variety of carbohydrates. There has been considerable fundamental and applied interest in the utilization of glucose and its alpha- and beta-polymers by hyperthermophiles. While glycolysis by Bacteria at high temperatures shows conventional characteristics, it has been found that glucose catabolism by hyperthermophilic Archaea differs from the canonical glycolytic pathways, involves novel enzymes, and shows a unique control. This review addresses these aspects with specific attention to Pyrococcus furiosus, which is one of the best studied hyperthermophilic Archaea, has the capacity to grow on a variety of sugars including the marine beta-(1,3)-linked glucose polymer laminarin, and has been found to contain three novel glycolytic enzymes, two ADP-dependent kinases, and a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase.

Receptor binding and internalization of a water-soluble (1-->3)-beta-D-glucan biologic response modifier in two monocyte/macrophage cell lines.

Abstract Glucan phosphate, a water-soluble, chemically defined (1-->3)-beta-D-glucan biologic response modifier, has been reported to exert antisepsis activity and accelerate wound healing. In this study we describe the specific binding of glucan phosphate to human and murine monocyte/macrophage cell lines, U937 and J774A.1, respectively. At 37 degrees C, equilibrium binding was rapidly achieved, i.e., within 1 min. In U937 cells, binding occurred with an affinity (Kd) of 37 microM and a Bmax of 65 x 106 binding sites/cell at 37 degrees C. In J774A.1 cells, glucan phosphate bound with an affinity (Kd) of 24 microM and a Bmax of 53 x 106 binding sites/cell at 37 degrees C. In both cases there was insignificant nonspecific binding. We further demonstrated that bound glucan phosphate cannot be displaced by a 50-fold excess of unlabeled ligand, suggesting internalization of glucan phosphate. Transmission electron microscopy showed significantly increased cytoplasmic vacuolization and significantly decreased mitotic activity in glucan phosphate-treated U937 cells compared with that in untreated cells. Pullulan, a random coil alpha-(1-->4)-(1-->6)-linked glucose polymer that served as a control, did not compete for the same binding site as glucan phosphate in either cell line, indicating the specificity of the binding site for (1-->3)-beta-D-glucans. We conclude that water-soluble pharmaceutical grade (1-->3)-beta-D-glucan phosphate specifically binds to and is internalized by U937 and J774A.1 cells.

Relationship of chemical shift to glycosidic conformation in the solid-state 13C NMR spectra of (1 → 4)-linked glucose polymers and oligomers: anomeric and related effects

Relationship of chemical shift to glycosidic conformation in the solid-state 13C NMR spectra of (1 → 4)-linked glucose polymers and oligomers: anomeric and related effects

Kinetics of Icodextrin

Icodextrin isanα-1,4 linked glucose polymer produced by hydrolysis of starch and membrane fractionation to obtain material with the desired molecular weight distribution. Icodextrin is similar in structure to physiological carbohydrates such as glycogen, but the latter has a higher degree of cross-linking through α-1,6 bonds. Icodextrin is a substrate for plasma α-amylase which hydrolyzes it to maltose, maltotriose, and maltotetrose. The fate of icodextrin administered in solution into the peritoneal cavity of patients in renal failure is determined by its stability in the peritoneal fluid, the extent of absorption, probably via the lymphatics, into the systemic circulation, and the rate of hydrolysis by plasma amylase. Early studies on a glucose polymer very similar to icodextrin demonstrated that the compounds are stable in peritoneal fluid from continuous ambulatory peritoneal dialysis (CAPD) patients, but rapidly hydrolyzed by plasma. Absorption of polymer from the peritoneal cavity was approximately 28% during a 12-hour dwell. In these studies it was apparent that hydrolysis products of up to four glucose units readily passed from the blood to the dialysis fluid, but higher molecular weight fragments wereexcluded. In the MIDAS study in which patients were treated with a single daily exchange of 2 L of 7.5% icodextrin, plasma samples were obtained during visit 2 (pretreatment with icodextrin), visit 6 (1 month after commencement of treatment), visit 9 (3 months), and visit 12 (6 months) and analyzed for icodextrin and metabolites. Steady-state plasma levels of icodextrin of approximately 4.6 mg/mL and maltose of 1.1 mg/mL were stable at 1,3, and 6 months. During special studies, when the dwell time for icodextrin and the comparator glucose solutions were controlled to 8 or 12 hours, dialysate was analyzed for polymer and metabolites. Total carbohydrate lost from the dialysate was approximately 19.6% after 8 hours and 33.5% after 12 hours. This compares with a mean of 85.6% of absorbed carbohydrate in 7 patients treated with strong glucose (6 on 3.86%, 1 on 2.27% glucose) for 8 hours. Plasma levels of icodextrin and maltose were measured in 12 patients who stopped once-a-day treatment with the polymer after approximately 2 years. Icodextrin (4.8 mg/mL) and maltose (1.1 mg/mL) fell to pretreatment levels in 7 10 days. On recommencement of treatment with polymer, plasma levels rose to steady-state levels of 4.7 mg/mL for icodextrin and 1.1 mg/mL for maltose in 7 -10 days. Icodextrin in solution in the peritoneal cavity is absorbed into the blood stream, probably via the lymphatic system. The extent of absorption depends on the dwell time of the solution; about 20% of total carbohydrate is absorbed in 8 hours, and this rises to 34% at 12 hours. There is no evidence of metabolism of glucose polymers like icodextrin in the peritoneal cavity. Icodextrin in the systemic circulation is hydrolyzed, probably by plasma and tissue α-amylase, to oligosaccharides, such as maltose, maltotriose and maltotetrose. These may be further metabolized, eliminated by peritoneal dialysis, or, if the patient has residual renal function, excreted in urine. The rapid attainment of steady-state plasma levels of icodextrin and its metabolites and the rapid decline when treatment is stopped is strong evidence against extensive tissue storage of the polymer.

The digestion of barley-based diets by cattle: the effect of feeding of three diets containing different amounts of formaldehyde-treated soybean meal

Six growing cattle, with intestinal fistulae, were fed on barley-based diets to which were added increments of formaldehyde-treated soybean meal; the latter was substituted for whole-grain barley. The digestion of organic matter (OM), �-linked glucose polymer (starch), nitrogen (N) and rumen volatile fatty acids (VFA) were measured at a restricted level of intake. The cattle were offered air-dried feed at 3.4 kg per head daily, or approximately ad libitum (i.e. 4.2 kg air-dried feed per head per day). The three diets contained 19.7, 27.8 or 42.9 g nitrogen (N) per kg and were fed in a Latin-square design. When the feed was offered ad libitum there was a significant increase in OM digestibility, from 78% on the lowest N diet to 84% on the highest N diet. Relatively large quantities of starch (140-477 g/day) entered and were apparently digested in the small intestine, especially on the 19.7 g N/kg dry matter (the highest starch diet) on ad libitum feeding. The molar proportions of VFA in rumen liquor largely reflected the amount of starch which was fermented in the rumen. The digestion of nitrogen was characterized by a net gain in flow to the duodenum compared with intake. There was a significant increase in the amount of N apparently digested in the small intestines on the 42.9 g N/kg dry matter diet. The proportion of microbial N at the duodenum declined as more formaldehyde-treated soybean meal was added to the diet. Compared with that of the lowest N diet, the amount of amino acids entering and being apparently digested in the small intestines doubled in the highest N diet, and the concentrations of plasma-free amino acids reflected this. The feed intake comparison indicated that differences in interpretations of digestion studies were likely if animals were fed solely at restricted intakes. The main discrepancies were in OM digestibility, starch digestion in the small intestine, the proportion of microbial N flow at the duodenum and in the estimation of amino acid absorption and requirement.

Comparative studies on β-glucan hydrolases: Isolation and characterization of an exo(1→3)-β-glucanase from the snail, Helix pomatia

An exo-β-glucan hydrolase, present in the digestive juice of the snail, Helix pomatia, has been purified to homogeneity by chromatography on Bio-Gel P-60, Sephadex G-200, DEAE-cellulose, and DEAE-Sephadex. The enzyme degrades β-(1 → 3)-linked oligosaccharides and polysaccharides, rapidly and to completion, or near completion, yielding glucose as the major product of enzyme action. Mixed linkage (1→3; 1→4)-β-glucans are also extensively degraded and β-(1→6)- and β-(1→4)-linked glucose polymers are slowly degraded by the enzyme. This enzyme differs from other exo-β-glucanases, reported previously, in the broadness of its substrate specificity. The K m values for action on laminarin and lichenin are respectively 1.22 and 2.22 mg/ml; the maximum velocity of action on laminarin is approximately twice that on lichenin. The enzyme has a molecular weight of 82,000 as determined by polyacrylamide gel electrophoresis. Maximum activity is exhibited at pH 4.3 and at temperatures of 50–55 °C.

The effect of H-ion concentration upon aerobic polysaccharide synthesis by resting cells of Saccharomyces cerevisiae.

A marked inhibition of polysaccharide synthesis by decreasing extracellular H-Ion Concentration has been shown. The decreased synthesis was found to be mainly associated with an inhibition of the synthesis of low molecular weight, trichloroacetic acid soluble, α1–4 linked glucose polymers.