Investigation of beta-glucans binding to human/mouse dectin-1 and associated immunomodulatory effects on two monocyte/macrophage cell lines

Dectin‐1, a specific pattern recognition receptor for β‐1,3/β‐1,6‐glucans, is expressed mainly on phagocytes. Human dectin‐1 (hDectin‐1) and mouse dectin‐1 (mDectin‐1) were separately expressed on HEK293 cell surfaces for examination of the binding abilities of a synthetic particulate β‐glucan (pβG), a product extracted from Saccharomyces cerevisiae, in this study. The binding of zymosan‐FITC to hDectin‐1 and mDectin‐1 was inhibited by pβG at similar concentrations for 50% inhibition of binding (IC₅₀). However, the kinetics of the time course and dose response to zymosan stimulation observed for U937 and J774A.1 differed. Superoxide anion production was increased in U937 but reduced in J774A.1 when cells were treated with pβG, zymosan, or laminarin, whereas ovalbumin endocytosis was enhanced in U937 and J774A.1 treated either with pβG, zymosan, laminarin, or barley‐glucan. These results indicate that the binding affinity of pβG to hDectin‐1 is similar to the binding affinity to mDectin‐1, and that stimulation by pβG as well as various forms of β‐1,3‐glucans on U937 and J774A.1 resulted in upregulation of cell activity and ovalbumin endocytosis. Additionally, other coreceptors on U937 and J774A.1 may be involved in directing different responses to superoxide anion production in these two types of cells. These results will likely contribute to further investigations on identifying the biological forms of β‐glucans capable of binding its specific receptor as the effective immunomodulators. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions

Thermal stabilization of the complex between polycytidylic acid [poly(C)] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the T(m) value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14 degrees C. In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm(-3)). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions.     

Separation of membranes from semiprotoplasts of suspension-cultured sycamore maple (Acer pseudoplatanus) cells

Semiprotoplasts were produced from suspension-cultured Acer pseudoplatanus (sycamore maple) cells prior to cell disruption by passing them through a 60 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on linear sucrose density gradients. Samples were collected by gradient fractionation and subcellular fractions were assayed for membrane markers and glycosyl transferase activities. Results of standard marker assays (cytochrome c reductase for endoplas-mic reticulum. uridine and inosine diphosphatases for Golgi. and eosin-5'-maleimide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of ¹⁴C-labeled sugars from UDP-glucose and UDP-xylose into ethanol precipitated polysaccharides was used to detect glucan synthases I & II (glucosyl transferases) and xylosyl transferase activities in Golgi membrane fractions. All three glycosyl transferases overlapped in fractions corresponding to both Golgi and plasma membrane markers, although peak activities for all three occurred in different fractions. More than one peak of glucan synthase I activity was found. Glucan synthase II, associated with ß-l.3 glucan (cullose) synthesis in plasma membranes, was also detected and exhibited a 10-fold stimulation in the presence of Ca²⁺.     

Short Note: Effect of Acremonium obclavatum glucan on groundnut leaf rust disease caused by Puccinia arachidis Speg.

A water soluble glucan purified from the culture filtrate of Acremonium obclavatum, an antagonist to the groundnut rust, Puccinia arachidis, inhibited germination of uredospores of P. arachidis. Prior treatment of groundnut leaves with glucan prolonged the incubation period between inoculation and development of rust disease, and caused a decrease in the number of pustules and uredospores/sorus. Treated groundnut leaves showed an increase in endogenous salicylic acid, intercellular chitinase and -1,3 glucanase activities.     

Design,characterization, and in vitro antiproliferative efficacy of gemcitabine conjugates based on carboxymethyl glucan

Gemcitabine (GEM) is widely used in clinical practice in the treatment of cancer and several other solid tumors. Nevertheless, the antitumor effect of GEM is partially prevented by some limitations including short half life, and lack of tumor localizing. Carboxymethyl glucan (CMG), a carboxymethylated derivative of β-(1-3)-glucan, shows biocompatibility and biodegradability as well as a potential anticarcinogenic effect. To enhance the antiproliferative activity of GEM, four water soluble conjugates of GEM bound to CMG via diverse amino acid linkers were designed and synthesized. ¹H NMR, FT IR, elementary analysis and RP-HPLC chromatography were employed to verify the correct achievement of the conjugates. In vitro release study indicated that conjugates presented slower release in physiological buffer (pH 7.4) than acidic buffer (pH 5.5) mimicking the acidic tumor microenvironment. Moreover, A549, HeLa and Caco-2 cancer cell lines were used to evaluate the in vitro cytotoxicity of conjugates and the results showed that binding GEM to CMG significantly enhanced antiproliferative activity of GEM on A549 cells. Therefore, these conjugates may be potentially useful as a delivery vehicle in cancer therapy and worthy of further study on structure-activity relationship and antiproliferative activity in vitro and in vivo, especially for lung tumor.     

Insolubilization of exogenous primer dextran with 1,3-α-d-glucan synthase from Streptococcus mutans

Abstract The water-insolubilization mechanism of exogenous primer dextran with 1,3-α- d -glucan synthase (EC 2.4.1.-) from Streptococcus mutans was studied. The 1,3-α- d -glucan synthase solution, containing sucrose and exogenous primer dextran, was incubated briefly. Water-insoluble glucan was synthesized. At the same time, water-soluble glucan, mainly derived from exogenous primer dextran, decreased. Linkage analysis data of glucan produced revealed that 1,3-α- d -glucoside bonds increased. Exogenous primer dextran was changed by the action of 1,3-α- d -glucan synthase to water-insoluble glucan. The results suggest that in a short-term reaction system of outside primer-insertion type, the 1,6-α- d -glucoside bond forms the main chain of water-insoluble glucan.     

赤芝孢子粉葡聚糖LB-NB的结构与构象(英文)

从破壁赤芝孢子粉的碱提粗多糖中分离纯化得到一个新的葡聚糖 ,命名为LB NB ,Mr为 4 .7× 1 0 4 ,[α]2 1D - 2 4 .52 0(c 0 .81 ,H2 O)。通过核磁共振、全水解、甲基化反应和Smith降解确定其结构为 β D ( 1→ 3)葡聚糖 ,每 4 .4个糖残基的 6位接有单一的端基葡萄糖。根据不同NaOH浓度下旋光度 [α]D 及特性粘度 [η]的变化 ;H2 O Me2 SO体系中特性粘度 [η]和Hug gins常数k′的变化及刚果红实验 ,推测LB NB在水及低浓度NaOH溶液中 ( <0 .0 5mol/L)呈单螺旋构象 ,而在Me2 SO及高NaOH浓度溶液中 ( >0 .1mol/L)中以无规则卷曲结构存在。体外免疫活性筛选表明 ,LB NB能显著促进T细胞的分化增殖 ,但对B细胞无明显作用     

Plant and algal cell walls: diversity and functionality

Background

Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed.

Scope

The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research.     

Arrangement of mixed-linkage glucan and glucuronoarabinoxylan in the cell walls of growing maize roots

Background and Aims

Plant cell enlargement is unambiguously coupled to changes in cell wall architecture, and as such various studies have examined the modification of the proportions and structures of glucuronoarabinoxylan and mixed-linkage glucan in the course of cell elongation in grasses. However, there is still no clear understanding of the mutual arrangement of these matrix polymers with cellulose microfibrils and of the modification of this architecture during cell growth. This study aimed to determine the correspondence between the fine structure of grass cell walls and the course of the elongation process in roots of maize (Zea mays).

Methods

Enzymatic hydrolysis followed by biochemical analysis of derivatives was coupled with immunohistochemical detection of cell wall epitopes at different stages of cell development in a series of maize root zones.

Key Results

Two xylan-directed antibodies (LM11 and ABX) have distinct patterns of primary cell wall labelling in cross-sections of growing maize roots. The LM11 epitopes were masked by mixed-linkage glucan and were revealed only after lichenase treatment. They could be removed from the section by xylanase treatment. Accessibility of ABX epitopes was not affected by the lichenase treatment. Xylanase treatment released only part of the cell wall glucuronoarabinoxylan and produced two types of products: high-substituted (released in polymeric form) and low-substituted (released as low-molecular-mass fragments). The amount of the latter was highly correlated with the amount of mixed-linkage glucan.

Conclusions

Three domains of glucuronoarabinoxylan were determined: one separating cellulose microfibrils, one interacting with them and a middle domain between the two, which links them. The middle domain is masked by the mixed-linkage glucan. A model is proposed in which the mixed-linkage glucan serves as a gel-like filler of the space between the separating domain of the glucuronoarabinoxylan and the cellulose microfibrils. Space for glucan is provided along the middle domain, the proportion of which increases during cell elongation.