A lectin histochemical study of the oesophagus of shi drum

The saccharide composition of surface and secretion glycoconjugates in the oesophagus of Umbrina cirrosa was examined by means of lectin histochemistry. Mucous cells showed the presence of N‐acetylgalactosamine, N‐acetylglucosamine and sialic acid linked to the dimer galactosyl(β1→3) N‐acetylgalactosamine. Columnar epithelial cells had a positive reaction with almost all the lectins employed, located in the supranuclear region and in the cell coat. The presence of abundant and various glycoconjugates in the secretions of shi drum oesophagus was correlated to the absence of salivary glands in fishes in general.     

Histology and carbohydrate histochemistry of the alimentary canal in the rainbow trout Oncorhynchus mykiss

A histological and histochemical analyses were carried out on the entire alimentary canal of the rainbow trout Oncorhynchus mykiss . In particular the oesophageal region showed presence of terminal β‐D‐galactose(1–3)‐N‐acetylgalactosamine and α‐N‐acetylgalactosamine. In the anterior and posterior regions of the stomach, lining epithelium and gastric pits exhibited the presence of β‐gal and α‐GalNAc. In addition sialoglycoconjugates having sialic acid–β–galactose(1–3)‐N‐acetylgalactosamine and sialic acid‐N‐acetylgalactosamine as terminal tri‐ and di‐saccharides, were demonstrated. In proximal and distal intestine goblet cells showed the presence of sialoglyconjugates, having sialic acid‐β‐gal(1–3)‐GalNAc and sialic acid‐GalNAc as terminal sequences, belonging to N‐linked chains. In the enterocytes of the entire intestine, terminal GlcNAc, α‐Gal, α‐fucose were found.     

Glycoconjugate characterization in the intestine of Umbrina cirrosa by means of lectin histochemistry

Goblet cells in the intestine of shi drum Umbrina cirrosa showed the presence of glycoconjugates particularly rich in fucose and N-acetylglucosamine residues. They displayed also sialic acid linked to galactosyl(β1→3)N-acetylgalactosamine and to galactosyl(β1→4)N-acetylglucosamine. All the nine horseradish peroxidase-conjugated lectins employed with the only exception of GSA II marked the enterocytes supranuclear region and the cell coat; the cell coat showed a more intense reactivity toward the different lectins, particularly enhanced with the use of fucosyl specific lectins.     

SPHINGOGLYCOLIPIDS IN THE NERVOUS SYSTEM IN FABRY'S DISEASE

Abstract— Two glycolipids, accumulated in the spinal ganglia of a patient with Fabry's disease were identified as: galactosyl (α1 → 4) galactosyl (β1 → 4) glucosyl(1 → 1) ceramide (CTH) and galactosyl (α1 → 4) galactosyl(1 → 1) ceramide (CDG). Only one glycolipid which had the same structure as the CTH in the spinal ganglia accumulated in the sympathetic ganglia of the patient. In the nervous system, CTH contained behenic acid (C_22:0) as the major fatty acid. In the spinal ganglia, CDG also contained behenic acid as the major fatty acid.     

A β-(1→4)-xylosyltransferase involved in the synthesis of arabinoxylans in developing barley endosperms

A β-(1→4)-xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley ( Hordeum vulgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5'-diphosphoxylose (UDP-Xyl) into exogenous β-(1→4)-xylooligosaccharides derivatized at their reducing ends with 2-aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β-(1→4)-xylotriose (Xyl₃-PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl₄₋₈-PA. Methylation analysis and enzymatic digestions with β-xylosidase (EC 3.2.1.37) and endo -β-(1→4)-xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β-(1→4)-linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X-100 and 5 m M MnCl₂. The apparent Michaelis constant and maximal velocity of the enzyme for Xyl₃-PA were 2.1 m M and 25 400 pmol min⁻¹ mg protein⁻¹, respectively. The enzyme also transferred [¹⁴C]Xyl from UDP-[¹⁴C]Xyl into higher β-(1→4)-xylooligosaccharides and birchwood xylans through β-(1→4)-linkages. The enzyme activity varied according to the stage of development (7–35 days after flowering) of the endosperms. Maximal activity occurred at 13–16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11–22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.     

Glycosides from Roots of Cyathula officinalis Kuan

Abstract: To search for new and bioactive compounds from traditional Chinese medicines, a new glycoside, 3-O-[α- L -rhamnopyranosyl-(1→3)-( n -butyl-β- D -glucopyranosiduronate)]-28-O-β- D -glucopyranosyloleanolic acid ( 1 ), was isolated from the roots of Cyathula officinalis Kuan, along with 3-O-(methyl-β- D -glucopyranosiduronate)-28-O-β- D -glucopyranosyl oleanolic acid ( 2 ), 3-O-β- D -glucopyranosyl oleanolic acid ( 3 ), 3-O-β- D -glucuronopyranosyl oleanolic acid ( 4 ), 3-O-[β- L -rhamnopyranosyl-(1→3)-(β- D -glucuronopyranosyl)] oleanolic acid ( 5 ), 3-O-(β- D -glucuronopyranosyl)-28-O-β- D -glucopyranosyl oleanolic acid ( 6 ), 28-O-β- D -glucuronopyranosyl-(1→4)-β- D -glucopyranosyl hederagenin ( 7 ), 3-O-[β- L -rhamnopyranosyl-(1→3)-β- D -glucuronopyranosyl]-28-O-β- D -glucopyranosyl oleanolic acid ( 8 ), and 3-O-[β- D -glucopyranosyl-(1→2)-α- L -rhamnopyranosyl-(1→3)-β- D -glucuronopyranosyl]-28-O-β- D -glucopyranosyl oleanolic acid ( 9 ). The structures of these compounds were determined based on spectral and chemical evidence. The 50 per cent growth-inhibiting (GI₅₀) of compounds 1 and 5 against MDA-MB-231 (a human breast cancer cell line) was 3.44 × 10^-4 and 4.66 × 10^-4 mol/L, respectively.
(Managing editor: Wei WANG)     

Coleoptile growth-inducing capacities of exo-β-(1→3)-glucanases from fungi

An exo-β-(1β3)-glucanase derived from Selerotinia libertiana induced growth of Avena sativa coleoptiles and degraded hemicelluloses and β-(1→4):(1→3) mixed linked glucan. However, neither endo-β-(1→4)- nor endo-β-(1→3)-glucanase activity could be detected in the enzyme preparation. Nojirimycin inhibited the glucan degradation caused by the enzyme but glucono-1,5-lactone did not. Another exo-β-(1→3)-glucanase derived from Basidiomycete QM 806 did not induce coleoptile growth and did not degrade the glucan. Growth-inducing properties of exo-β-(1→3)-glucanases are discussed.     

Use of Horseradish Peroxidase Conjugated Lectins for Detection of Glycoconjugate Changes in Developing Lingual Epithelium of the Chick Embryo

A battery of six different horseradish peroxidase-labelled lectins (SBA, DBA, PNA, WGA, ConA and LTA) was used to study the distribution of carbohydrate residues in glycoconjugates during the development of the chick embryo tongue anlage (7th-21st day of incubation) and in the tongue of 3 day-old chicken, at the dorsal and ventral epithelium. From the 7th to the 16th day β-D-galactose, D-galactose-(β1 → 3)-N-acetyl-D-galactosamine, D-mannose and β-N-acetyl-D-glucosamine show the same epithelial distribution at two lingual aspects. From the 17th day to hatching the epithelium of the dorsal and ventral surfaces is characterized by a different distribution of sugar residues. The functional adaptation of the epithelium, as far as the saccharidic moletie distribution is concerned, seems to be completed at the 21st day of incubation.     

The ftsQ1p gearbox promoter of Escherichia coli is a major sigma S-dependent promoter in the ddlB–ftsA region

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 ( Freese and Fortnagel, 1967 ) using PCR. After cloning and expression in E. coli , SDS–PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol, 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol and 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[ O -β- D -glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as ¹⁴C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-β- D -glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyldiacylglycerol or triglucosyldiacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in β (1→6) linkage to the product of the preceding reaction.     

Comparative study of extracellular and intracellular β-glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β-glucosidases of this yeast were purified by ion-exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β-glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β-glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)-β, (1 → 6)-β and (1 → 4)-α linkage configuration. The intracellular enzyme possesses (1 → 6)-α-arabinofuranosidase activity and extracellular enzyme (1 → 6)-α-rhamno-pyranosidase activity. The two β-glucosidases are competitively inhibited by glucose and by D-gluconic-acid-lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β-glucosidases for the liberation of the bound aroma is discussed.     

Proteolytic inactivation of an extracellular (1 → 3)-β-glucanase from the fungus Acremonium persicinum is associated with growth at neutral or alkaline medium pH

Abstract The filamentous fungus Acremonium persicinum released high levels of proteolytic enzyme activity into the culture fluid during growth at pH 7 or above. Almost total inhibition of this crude activity by phenylmethylsulfonyl fluoride suggested that it was mainly due to the presence of a serine protease. This protease inactivated one of three extracellular (1 → 3)- β -glucanases produced by this fungus, although the activities of the remaining two (1 → 3)- β -glucanases did not appear to be affected. Growth of A. persicinum in acidic conditions resulted in the presence of much lower extracellular proteolytic activity and no apparent (1 → 3)- β -glucanase inactivation.     

Release of sex steroids into the water by roach

Electro‐olfactogram (EOG) recordings of the olfactory epithelium of both male and female roach Rutilus rutilus demonstrated that both sexes were able to detect free and glucuronidated 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) with high sensitivity. Male, but not female, roach were also sensitive to androstenedione. Sexually mature female roach were shown to release free 17,20β‐P, glucuronidated 17,20β‐P and androstenedione into the water; for all three steroids, the rate of release was significantly enhanced by injection of carp pituitary extract (CPE). A series of trials was also carried out which showed that mature males, and to a lesser extent immature males and females, were able also to release free and glucuronidated 17,20β‐P, both before and after CPE treatment. Water extracts from containers that had held CPE‐treated mature male and female roach were examined for the presence of other steroids. This revealed that free and glucuronidated 17,20β‐P plus free and glucuronidated 17,20β,21‐trihydroxy‐4‐pregnen‐3‐one (17,20β, 21‐P) predominated in water extracts from both sexes. The free moieties of 17,20α‐dihydroxy‐4‐pregnen‐3‐one, 17‐hydroxyprogesterone and 11‐deoxycortisol were found at concentrations which were between four and 20 times lower than those of free 17,20β‐P. Androstenedione was found at concentrations which were 25‐fold lower than those of 17,20β‐P. Despite its apparent high rate of release by sexually mature male and female roach, free 17,20β,21‐P was found not to exhibit any EOG activity at the highest dose tested (10⁻⁷ M).     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

A New Steroidal Glycoside from Ophiopogon japonicus （Thunb.） Ker-Gawl.

To study the chemical constituents from traditional Chinese herb Ophiopogon japonicus （Thunb.） Ker-Gawl., a new steroidal glycoside, named ophiopojaponin C （1）, together with two known ones, was isolated by column chromatography. Spectroscopic and chemical evidence revealed the structures to be ophiopogenin 3-O-[α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→4）-β-D-glucopyranoside （1）, diosgenin 3-O-[2-O-acetyl-α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→3）-β-D-glucopyranoside （2）, and ruscogenin 1-O-[2-O-acetyl-α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→3）-β-D-fucopyranoside （3）.     

Glycoprotein Properties of the Solubilized Atrial Muscarinic Acetylcholine Receptor

Abstract: The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase-treated and untreated receptor suggest that high-affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N -acetylglucosamine or its β(1→4)-linked oligomers.     

Specificity of sialytransferase: structure of alpha1-acid glycoprotein sialylated in vitro. A new methodology for the determination of the structure of the linkage formed by glycosyltransferase action on galactosyl-oligosaccharide-protein acceptors.

The terminal galactosyl units of desialylated alpha1-acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H-labeled glycoprotein was effective as an acceptor in sialytransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP-N-acetyl-neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H-labeled glycoprotein yielded radioactive 2,3,4-trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C-6 of the terminal galactosyl units of desialylated alpha1-acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions. This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native alpha1-acid glycoprotein.     

Antibodies as probes for the study of location and metabolism of (1→3), (1→4)-β-D-glucans

Polyclonal antibodies, raised against ((1→3), (1→4)-β-D-glucans from oat ( Avena sativa L.) caryopsis, were used to investigate the location and the metabolism of mixed-linked β-D-glucans. The binding of these antibodies to the cell walls of oat coleoptiles was shown by an indirect fluorescence method. Distinct fluorescent regions were observed along the inner layers of the walls of each cell. The preimmune serum or antibodies pretreated with oat caryopsis β-D-glucans did not react with the cell walls. Glucan antibodies were bound to the walls of other Poaceae coleoptiles as well as to those from oat mesocotyls and roots, whereas they were not bound to the walls of some dicotyledons tested. The relative glucan content of the cell walls of oat coleoptiles as determined by β-D-glucanase (EC 3.2.1.73) treatment was maximum between day 3 and 4 after soaking, but it declined during further elongation. A rapid decrease in glucan content was observed in excised coleoptiles when auxin or β-D-glucanase was present. There was a clear correlation between the glucan content expressed on a basis of cell wall polysaccharides and the amount of the antibodies bound to the cell walls. These results indicate that the antibodies are useful probes to detect and determine (1→3), (1→4)-β-D-glucans of cell walls.     

Purification and further characterization of a glycoprotein from the skin mucus of Japanese eels

A glycoprotein (EGP) was purified from the skin mucus of Japanese eel Anguilla japonica . Apparent average molecular mass of the EGP was estimated to be 500 000. The EGP was found to contain 30·8% NeuAc, 26·4% GalNAc, 6·4% Gal, 0·4% NeuGc and 25·1% Thr‐rich protein. EGP was treated with alkaline borohydride for the release of carbohydrate chains (oligosaccharide alditols). Three carbohydrate chains were isolated from the released carbohydrate chains by Sephadex G‐25 (superfine) gel filtration and HPLC. Using ¹H‐NMR spectroscopy, methylation analysis and glycosidase digestion, the structures of the three carbohydrate chains were determined to be NeuAcα2→6GalNAc‐ ol , NeuAcα2→3GalNAc‐ ol and NeuAcα2→6(GalNAcα1→3)GalNAc‐ ol . An overall structure for the sialoglycoprotein from the skin mucus is proposed: the molecule is considered to consist of highly glycosylated 10 glycopeptide units (containing >40 carbohydrate chains) that are linked to the hydroxyl amino acids and spaced on average two amino acids apart.     

Determination of specificities of rabbit antisera against the O-antigenic polysaccharide from Escherichia coli O126

Abstract Rabbit polyclonal antibodies against the lipopolysaccharide of Escherichia coli O126 were serologically characterized by ELISA. The antibody specificities were determined by studying the inhibitory effects of the methyl glycosides of both anomeric configurations of the constituent monosaccharides and the oligosaccharides derived from the O-antigenic polysaccharides of E. coli O126. It was found that, amongst the monosaccharides, β- d -N-acetyl glucosamine was the most effective inhibitory sugar in the O126 polysaccharide and the major specificity of the polyclonal antibodies was found to be directed against the trisaccharide having the structure α- D -Gal p (1 → 3)-β- D -Glc pNAc(1→2)- D -Man p.     

Study of the glycosylation of apolipoprotein H

Apolipoprotein H is a single chain polypeptide composed of 326 amino acids highly glycosylated. Its carbohydrate content is approximately 19% of the molecular weight. We show that it is rich in sialic acid linked alpha (2-6) to galactose or N-acetylgalactosamine. Sialic acid is not alpha (2-3) linked to galactose. Galactose is beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to N-acetylgalactosamine. Carbohydrate O-linked chains (mainly sialic acid) are alpha (2-6) linked to galactose or N-acetylgalactosamine. Galactose is also organised in O-linked chains and beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to acetylgalactosamine. Concanavalin A lectin was used to isolate two groups of apolipoprotein H molecules bearing biantennary and truncated hybrids and high mannose and hybrid oligosaccharides. Apolipoprotein H fails to bind lysine-Sepharose. Our results thus show that it presents truncated hybrid or hybrid-type carbohydrate chains which bear few unmasked mannose residues as a terminal sugar. Biochemical analysis of carbohydrate structures conducted on single isoforms separated through IEF revealed that no specific carbohydrate complex is bound to a single isoform.