Prospects for the Practical Application of Substrate-Binding Modules of Glycosyl Hydrolases

The properties of substrate-binding modules of glycosyl hydrolases have been reviewed. The variation of the properties of these modules makes them promising as components of chimeric proteins, which is a rapidly developing field of biotechnology. Examples of applying substrate-binding modules of glycosyl hydrolases to the immobilization of proteins and whole cells on polysaccharides and the purification of proteins are described. Promising methods for (1) detecting various compounds using hybrids of substrate-binding modules with antibodies and (2) locating polysaccharides in live tissues are reviewed as well.     

Prospects for practical application of substrate-binding modules of glycosyl hydrolases (A review)

The properties of substrate-binding modules of glycosyl hydrolases have been reviewed. Variation of the properties of these modules makes them promising as components of chimeric proteins, which is a rapidly developing field of biotechnology. Examples of applying substrate-binding modules of glycosyl hydrolases to immobilization of proteins and whole cells on polysaccharides and purification of proteins are described. Promising methods for (1) detecting various compounds using hybrids of substrate-binding modules with antibodies and (2) locating polysaccharides in live tissues are reviewed as well.     

Isolation, partial characterization, and biological properties of polysaccharides from crude papain

Two polysaccharides have been isolated from crude papain by precipitation of papain with ammonium sulfate, further precipitation of other proteins with trichloroacetic acid, and chromatography of the supernatant on DEAE-cellulose. The first polysaccharide to be eluted, designated PP-I, contained D-glucuronic acid, D-glucose, D-galactose, L-arabinose, and L-rhamnose, in the approximate molar ratios of 4:1:12:10:4. The other (PP-II), eluted at a higher salt-concentration, contained the same sugars (with about one-third less glucose and more uronic acid) in the approximate molar ratios of 13:1:40:26:12. Reduction of the uronic acid groups of PP-II produced a polysaccharide (PP-II-R) containing the same sugars in the approximate molar ratios of 2:11:37:28:12. Hydrolysis of a mixture of the two polysaccharides yielded an aldobiouronic acid, D-glucosyluronic acid-D-galactose. Neither polysaccharide preparation contained protein. These polysaccharides dramatically affected aggregation and alignment of normal human fibroblasts but had no effect on a mouse embryo fibroblast aneuploid cell-line that does not exhibit contact inhibition of growth or movement. In aggregating cells, these polysaccharides caused the cells to behave as contact-inhibited cells, that is, cell division and nuclear area were decreased.     

Distribution of insoluble polysaccharides, neutral lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. (Bignoniaceae)

In this study, distribution of polysaccharides, lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. was examined from sporogenous cell stage to mature pollen, using cytochemical methods. To detect the distribution and dynamic changes of insoluble polysaccharides, lipid bodies, and proteins in the anthers through progressive developmental stages, semi-thin sections of anthers at different developmental stages were stained with periodic-acid-Schiff (PAS) reagent, Sudan black B, and Coomassie brilliant blue, respectively, and examined under light microscope. Ultrastructural observations with TEM were also carried out to determine the storage form of starch in the connective tissue, and storage form of lipids in the tapetal cells. In sporogenous cell stage, anther wall contains numerous insoluble polysaccharides. However, from the sporogenous cell stage to the vacuolated microspore stage, the amount of insoluble polysaccharides in the anther wall decreases gradually. At bicellular pollen stage, tapetum degenerates completely and polysaccharides are not seen in the anther wall. Lipid bodies are observed in the cytoplasm of both middle layer and tapetal cells at tetrad stage, whereas they disappear in the vacuolated microspore stage. Compared with polysaccharides, proteins are limited in the anther wall at early stages of development. During pollen development, polysaccharides, proteins, and lipid bodies are scarce in the cytoplasm of sporogenous cells, but their amount increases at premeiotic stage. From tetrad stage to bicellular pollen stage, microspore cytoplasm contains variable amount of insoluble polysaccharide grains, lipid and protein bodies. At bicellular pollen stage, plentiful amount of starch granules are stored in the cytoplasm of the pollen grains. Proteins and lipid bodies are also present in the cytoplasm.     

Histochemistry of the glands associated with the reproductive tract ofLymnaea stagnalis

Summary The albumen gland, the muciparous gland and the oöthecal gland of female genital tract of Lymnaea stagnalis, collected in spring, autumn and winter have been studied.The reactions for polysaccharides, proteins and RNA have been performed in order to characterise the secretion of the glands.The albumen gland secretion consists almost exclusively of slightly acid polysaccharides whose histochemical reactions, according to Lison and Grainger and Shillitoe confirm the presence of galactogen. Proteins are also present in the secretion. The muciparous gland secretion consists of strongly acid mucopolysaccharides (non sulphated) produced by large cells among which small cells containing sulphated mucopolysaccharides are present.In the oöthecal gland two zones are present, one with a single type of cells containing acid mucopolysaccharides, and the other with two different types of cells: the first with mucopolysaccharides and the second with sulphated mucopolysaccharides, proteins and glycogen at the basis of the cell. Sialic acids are not present in the secretion of the glands studied.The polysaccharidic composition of the secretion of the glands is different from gland to gland. The secretion of the glands gradually changes and gets acid according to the composition of the various membranes and envelopes wrapping up the eggs.     

The role of cellular polysaccharides in the formation and stability of aerobic granules

AIMS: This paper attempts to investigate the role of cellular polysaccharides in the formation and stability of aerobic granules. METHODS AND RESULTS: Three column sequential aerobic sludge blanket reactors (R1, R2 and R3) were operated at a superficial air upflow velocity of 0.3 cm s(-1), 1.2 cm s(-1) and 2.4 cm s(-1), respectively. Aerobic granules appeared at cycle 42 in R2 and R3 with a mean size of 0.37 mm in R2 and 0.35 mm in R3, however, aerobic granulation was not observed in R1. After the formation of aerobic granules, the sludge volume index (SVI) decreased to 55 ml g(-1) in R2 and 46 ml g(-1) in R3. Aerobic granulation was concurrent with a sharp increase of cellular polysaccharides normalized to cellular proteins, which increased from 5.7 to 13.0 mg per mg proteins in R2, and 7.5-13.9 mg per mg protein in R3. The content of polysaccharides in aerobic granules was 2-3 times higher than that in the bioflocci cultivated in R1. The disappearance of aerobic granules in R2 was tightly coupled to a drop in cellular polysaccharides. After the reappearance of bioflocci in R2, the content of cellular polysaccharides were found to be restored to the level observed in R1. CONCLUSION: It appears that the production of cellular polysaccharides could be stimulated by hydrodynamic shear force and contributes to the formation and stability of aerobic granules. SIGNIFICANCE AND IMPACT OF THE STUDY: It is expected that this study would provide useful information for better understanding the mechanisms of aerobic granulation.     

Characterization and immunomodulatory activities of sulfated polysaccharides from Capsosiphon fulvescens

Sulfated polysaccharides isolated from Capsosiphon fulvescens and fractionated using ion-exchange chromatography were investigated to determine their chemical and molecular characteristics and biological activities. The crude and fractionated polysaccharides (F(1), F(2), and F(3)) consisted mostly of carbohydrates (28.9-67.0%), uronic acids (1.6-9.2%) and sulfates (5.2-13.4%) with various amounts of proteins (2.1-53.7%). Their monosaccharide levels were significantly different including rhamnose (20.8-65.2%), xylose (13.0-37.1%) and mannose (11.6-65.1%). The polysaccharides contained one or two subfractions with molecular weights (M(w)) ranging from 401.7×10(3) to 6232×10(3)g/mol. These polysaccharides (the crude and fraction F(2)) strongly stimulated macrophage cells, RAW264.7 cell line, producing considerable amounts of NO, PGE(2) and cytokines which suggested that they could be strong immunostimulators. The main backbone of the most immunoenhancing polysaccharide (F(2)) was suggested by GC-MS and NMR to be the following:     

Plant cell enlargement and the action of expansins

Plant cells are caged within a distended polymeric network (the cell wall), which enlarges by a process of stress relaxation and slippage (creep) of the polysaccharides that make up the load-bearing network of the wall. Protein mediators of wall creep have recently been isolated and characterized. These proteins, called expansins, appear to disrupt the noncovalent adhesion of matrix polysaccharides to cellulose microfibrils, thereby permitting turgor-driven wall enlargement. Expansin activity is specifically expressed in the growing tissues of dicotyledons and monocotyledons. Sequence analysis of cDNAs indicates that expansins are novel proteins, without previously known functional motifs. Comparison of expansin cDNAs from cucumber, pea, Arabidopsis and rice shows that the proteins are highly conserved in size and amino acid sequence. Phylogenetic analysis of expansin sequences suggests that this multigene family diverged before the evolution of angiosperms. Speculation is presented about the role of this gene family in plant development and evolution.     

Amyloid peptides derived from CsgA and FapC modify the viscoelastic properties of biofilm model matrices

The bacterial biofilm is a complex environment of cells, which secrete a matrix made of various components, mainly polysaccharides and proteins. An understanding of the precise role of these components in the stability and dynamics of biofilm architecture would be a great advantage for the improvement of anti-biofilm strategies. Here, artificial biofilm matrices made of polysaccharides and auto-assembled peptides were designed, and the influence of bacterial amyloid proteins on the mechanical properties of the biofilm matrix was studied. The model polysaccharides methylcellulose and alginate and peptides derived from the amyloid proteins curli and FapC found in biofilms of Enterobacteriaceae and Pseudomonas, respectively, were used. Rheological measurements showed that the amyloid peptides do not prevent the gelation of the polysaccharides but influence deformation of the matrices under shear stress and modify the gel elastic response. Hence the secretion of amyloids could be for the biofilm a way of adapting to environmental changes.     

The fine structure of the cystogenic cells of the cercaria of Fasciola hepatica L.

Summary The wall of the cyst of the metacercaria of the liver fluke (Fasciola hepatica L). is composed of four layers: an external tanned protein, two layers giving reactions of proteins and polysaccharides and an internal, finely laminated layer of keratinized protein. Each of the precursors of these layers is synthesised in a distinct kind of cystogenic cell in the cercaria, while it is still within the redia in the intermediate host, a snail.The cells forming the protein precursors are similar in cytoplasmic structures to secretory cells such as those of the exocrine pancreas. The cells producing protein and polysaccharides resemble mucinogenic cells.The keratin precursor is a rodlet formed by the rolling of a sheet into a scroll and all stages of this process can be recognised in the synthesising cells in the early cercaria.     

Extracellular polymeric substances enhanced mass transfer of polycyclic aromatic hydrocarbons in the two-liquid-phase system for biodegradation

The objective was to elucidate the role of extracellular polymeric substances (EPS) in biodegradation of polycyclic aromatic hydrocarbons in two-liquid-phase system (TLPs). Therefore, biodegradation of phenanthrene (PHE) was conducted in a typical TLPs—silicone oil–water—with PHE-degrading bacteria capable of producing EPS, Sphingobium sp. PHE3 and Micrococcus sp. PHE9. The results showed that the presence of both strains enhanced mass transfer of PHE from silicone oil to water, and that biodegradation of PHE mainly occurred at the interfaces. The ratios of tightly bound (TB) proteins to TB polysaccharides kept almost constant, whereas the ratios of loosely bound (LB) proteins to LB polysaccharides increased during the biodegradation. Furthermore, polysaccharides led to increased PHE solubility in the bulk water, which resulted in an increased PHE mass transfer. Both LB-EPS and TB-EPS (proteins and polysaccharides) correlated with PHE mass transfer in silicone oil, indicating that both proteins and polysaccharides favored bacterial uptake of PHE at the interfaces. It could be concluded that EPS could facilitate microbial degradation of PHE in the TLPs.     

Molecular characteristics of sulfated polysaccharides from Monostroma nitidum and their in vitro anticancer and immunomodulatory activities

We investigated water-soluble sulfated polysaccharides isolated from Monostroma nitidum using ion-exchange chromatography to determine their molecular characteristics and biological activities. The crude and fractionated polysaccharides (F(1), F(2), and F(3)) consisted mostly of carbohydrates (58.3-91.9%), uronic acids (0-21.8%) and sulfates (1.8-17.7%) as well as varying amounts of proteins (1.6-9.4%). Their monosaccharide levels were significantly different including rhamnose (0-95.7%) and glucose (0-98.6%) content with small amounts of xylose (0.8-4.3%). These polysaccharides contained one or two subfractions with average molecular weights (M(w)) ranging from 94.4 to 1387×10(3) g/mol. The in vitro inhibitory activity (≤75%) of the polysaccharides on a human cancer cell line (AGS) suggested that the polysaccharides had direct cytotoxic effects on the cancer cells. In addition, these hetero-polysaccharides (from the crude and F(1) and F(2) fractions) stimulated a macrophage cell line, Raw 264.7 cells, inducing considerable NO and PGE(2), production, which suggested that they could be strong immunomodulators.     

Detection of antigenic surface proteins of Actinobacillus actinomycetemcomitans using the immunoblotting method

Antigenic surface proteins of Actinobacillus actinomycetemcomitans (three strains), which can be recognized by antibodies in human serum, were examined using the Western blot method. By comparing the immunoblotting profiles between protease-treated cells and untreated cells, IgG-antigenic and IgM-antigenic surface proteins were found. The IgG-antigenic proteins revealed the following molecular weights: strain ATCC 29522, 52 and 49 kD; strain ATCC 29523, 45, 49, 52 and 70 kD; strain Y4, 36, 38, 44, 53 and 58 kD. Molecular weights of the IgM-antigenic proteins ranged from 50 to 92 kD: strain ATCC 29522, 68, 80, 90 and 92 kD; strain ATCC 29523, 62, 68 and 80 kD; strain Y4, 50, 64, 73, 81 and 86 kD. The IgG-antigenic proteins were very sensitive to trypsin and Bacillus licheniformis protease, but were resistant to V8 protease, while the IgM-antigenic proteins were sensitive to various proteases. These results suggested that IgG-antigenic and IgM-antigenic components were different from the serotype-specific antigen or species-specific antigen associated with polysaccharides or lipopolysaccharides with respect to molecular weights and that they were proteins.     

Ultrastructural and histochemical study of the salivary glands of Aplysia depilans (Mollusca, Opisthobranchia)

The digestive system of the sea hare, Aplysia depilans , includes a pair of ribbon-shaped salivary glands. A central duct and a large blood vessel run close to each other along the length of these glands and both are surrounded by a layer of muscle cells. Three cell types form the glandular epithelium: granular cells, vacuolated cells and mucocytes. The granular cells possess cilia and spherical secretion granules, located primarily in the apical region. The granules of immature cells have a low electron density and are mainly formed by neutral polysaccharides with small amounts of proteins. The granules of mature cells are larger, have a high electron density and are mainly formed by proteins with lower amounts of neutral polysaccharides. Transition stages between immature and mature granular cells are observed. The vacuolated cells are large and frequently pyramidal in shape, but after the application of histochemical techniques almost all vacuoles remain uncoloured. The numerous vacuoles contain flocculent material in a clear background and the mitochondria possess large crystalline structures in the matrix. A pyramidal shape is also typical of the mucocytes, which are filled with vesicles containing granular masses surrounded by a network of secretion material. These large cells are strongly stained by Alcian blue, revealing the presence of acidic mucopolysaccharides. This is the first ultrastructural study of the salivary glands in opisthobranch gastropods.     

锗对霍山石斛类原球茎悬浮培养细胞生长和多糖合成及氧化还原态的影响

为解决霍山石斛类原球茎液体培养细胞生长缓慢和代谢水平低下的问题,研究了不同浓度的锗(GeO2)对霍山石斛类原球茎增殖、多糖合成及碳氮利用的影响,分析了类原球茎细胞内还原糖、可溶性蛋白质含量、抗氧化酶活性以及细胞氧化还原态的变化。结果表明,适当浓度的二氧化锗(4.0mg/L)显著促进霍山石斛类原球茎的增殖和多糖的积累,最大细胞干重为32.6g/L,最大多糖产量为3.78g/L;显著提高胞内还原糖和可溶性蛋白质含量,超氧化物歧化酶和过氧化氢酶的活性明显升高,而过氧化物酶的活性则有所降低;氧化还原态分析发现,二氧化锗处理的细胞内还原型谷胱甘肽/氧化型谷胱甘肽的值明显提高,谷胱甘肽还原酶活性升高。添加适量的二氧化锗有利于细胞生长和多糖合成。     

龙葵腺毛发育的细胞学研究

利用光学显微镜、扫描电镜和透射电镜技术,观察了龙葵“四叶一心”期时叶片及茎表皮的腺毛的种类、分布,探究了不同类型腺毛的起源、生长、成熟、分泌、衰老等发育过程的细胞学特征;通过组织化学染色和荧光显微技术,观察了龙葵腺毛成分、分布,为龙葵的进一步开发利用提供参考。结果表明：（1）龙葵腺毛分为单细胞头腺毛和多细胞头腺毛两类,前者主要分布于茎表面和叶上下表皮,后者主要分布于茎表面的单细胞头腺毛之间、叶脉及叶边缘;（2）龙葵腺毛发育起始于表皮细胞突起,单细胞头腺毛行顶端生长,具1-4个柄细胞,四种类型;多细胞头腺毛可再分为一层、两层与三层多细胞头腺毛,另具三种特殊类型;（3）龙葵成熟腺毛具分泌能力,通过皮下空间的物质积累导致腺毛头细胞表面形成突起、包块、破口,最终释放分泌物;而头细胞与柄细胞随即皱缩、衰老。（4）超微结构显示,腺毛头细胞中内质网与高尔基体极为丰富,合成代谢及分泌活动活跃,产生大量包裹嗜锇物质的囊泡,囊泡与细胞壁融合,进而将嗜锇物质转移至细胞壁并积累,随后储存在角质层下的皮下空间直至分泌释放;（5）组织化学染色结果表明,腺毛含有萜类、生物碱、脂类、蛋白质、酚类和多糖。头细胞中主要含有萜类、生物碱、脂类、蛋白质、酚类和中性多糖;柄细胞中主要含有萜类、生物碱、脂类。     

Concentration of the proteins of skimmed milk by membraneless,isobaric osmosis

The concentration of skimmed milk proteins by polysaccharides such as gum arabic, arabinogalactan and apple pectin with a high content of methoxyl groups was studied. Investigation of the thermodynamic compatibility of skimmed milk proteins with these polysaccharides at different NaCl concentrations and pH has shown that above a certain polysaccharide concentration termed the ‘threshold of complete incompatibility’ the protein is almost completely excluded from the polysaccharide phase. Phase diagrams obtained for the systems: water-skimmed milk proteins-arabinogalactan, water-skimmed milk proteins-gum arabic and water-skimmed milk proteins-pectin, indicate that highly esterified apple pectin is superior to the other polysaccharides for concentrating skimmed milk proteins.The proposed method of concentration which may be called ‘membraneless isobaric osmosis' has a higher productivity and lower energy consumption than other methods of biopolymer concentration.     

Tumor-like lesions in the mantle of the mussel Modiolus difficilis from the Sea of Japan

Two inner growths in the mantle beneath the epithelium were found in 1 of 1000 mussels Modiolus difficilis from Amursky Bay, Sea of Japan, within the city precincts of Vladivostok. Both growths were about 2000 microns in maximal diameter in section and elevated slightly above the mantle surface. The mantle epithelium near the growths formed deep invaginations, and clusters of mucous cells were numerous beneath the epithelium. Histological and histochemical methods were employed. Two different kinds of growth were revealed. The off-white growth consisted of cells with thin granular or vesicular cytoplasm containing glucosaminoglycans, proteins and a small amount of neutral polysaccharides. Growth cells were pure white in color after treatment of preparations with 1% H2SO4 and differed markedly from the mantle cells. The yellow growth consisted of large granular cells with neutral polysaccharides and proteins. Although growths were composed of different kinds of cells, they seemed to be derived from subepithelial mucous cells. This was supported by histological and histochemical staining reactions of some tumor and mantle epithelial cells. Mitotic indices (MI) of growths and subepithelial mucous cells were zero, MI of ciliated mantle epithelium reached 0.07%. The lesions were areas of strongly altered mucous cells of mantle epithelium and were non-neoplastic.     

The extraction process optimization and physicochemical properties of polysaccharides from the roots of Euphorbia fischeriana

Response surface methodology (RSM) was used to determine the optimum extraction conditions for polysaccharides (EFP) from the roots of Euphorbia fischeriana. A Box-Behnken design (BBD) with four independent variables was investigated, such as extraction temperature (°C), water/solid ratio, extraction number (n), and extraction time (h). The results indicated optimum extraction conditions were extraction temperature of 97 °C, water/solid ratio of 9:1, extraction number of 2 and extraction time of 2.4 h, respectively. Under these conditions, the experimental value was 24.6 ± 0.62, which was well in close agreement with value predicted by the model. The preliminary chemical analysis of EFP revealed the EFP contained 25.43% polysaccharides, 20.42% uronic acids, 2.54% sulfate radical and 23.41% proteins. And the neutral polysaccharides were mainly composed of glucose, arabinose, rhamnose, galactose, xylose, mannose in the ratio of 21:8:5:3:1:1.     

Effect of Brefeldin A on the synthesis and transport of cell wall polysaccharides and proteins in pea root seedlings

The in vivo effect of Brefeldin A (BFA) on the synthesis andtransport of cell wall polysaccharides and proteins in the rootsof pea seedlings (Pisum sativum L. cv. Alaska) was investigated.BFA (10µgml^—1 inhibited the synthesis of cell wallmatrix polysaccharides by approximately 43%. Under the sameconditions, cellulose synthesis was inhibited by approximately77%. The percentage of incorporation of L—[U—¹⁴C]leucineand L-[U-14C]proline into cytosolic, membrane and cell wallproteins was only slightly changed in the presence of BFA. Inaddition, the drug did not change the pattern of newly synthesizedproteins in the three fractions as judged by SDS—PAGEfluorography. Double labelling of proteins and cell wall polysaccharidesconfirmed the above reported data. All these results showedthat the synthesis and transport of proteins to the cell wallwas only slightly affected by BFA under similar conditions tothose which brought about a strong inhibition of the synthesisof matrix and cellulosic polysaccharides. BFA had no effecton the activity of membrane-bound and digitonin-solubilizedmannan and glucomannan synthase isolated from the third internodeof pea seedlings. This would exclude an effect of BFA at thelevel of the catalytic site of the synthases. The inhibitionof polysaccharide synthesis by the drug was rapidly eliminatedafter its removal. It is concluded that the effect of BFA onthe biosynthesis of cell wall polysaccharides could be causedby an interaction of the drug with the topological organizationof the synthase complexes in the membranes. This effect wouldprecede the action of the drug at the level of vesicle transportto the walls. Key words: Brefeldin A, cell wall polysaccharides (synthesis and transport), Pisum sativum L, polysaccharide synthases, proteins (synthesis and transport)