An acidic xylan from extracellular polysaccharides of suspension-cultured cells of Nicotiana tabacum

An acidic xylan was isolated from the extracellular polysaccharides of suspension-cultured tobacco cells. Its structure was investigated by methylation analysis and ¹³C NMR spectroscopy and was shown to consist of a main chain of β-(1→4)-linked D-xylopyranosyl residues to which were attached as side-chains, α-D-glucuronic acid residues at O-2.     

An arabinogalactan from the skin of Opuntia ficus-indica prickly pear fruits

The cold-water extract from the skin of Opuntia ficus-indica fruits was fractionated by anion-exchange chromatography. The major fraction, which was purified by size exclusion chromatography, consisted of a polysaccharide composed of galactose and arabinose residues in the ratio 6.3:3.3, with traces of rhamnose, xylose and glucose, but no uronic acid. The results of methylation analysis, supported by (13)C NMR spectroscopy, indicated that this polysaccharide corresponded to an arabinogalactan having a backbone of (1-->4)-linked beta-D-galactopyranosyl residues with 39.5% of these units branched at O-3. The side-groups consisted either of single L-arabinofuranosyl units or L-arabinofuranosyl alpha-(1-->5)-linked disaccharides. This polysaccharide is thus an arabinogalactan that can be classified in the type I of the arabinogalactan family.     

Structural investigations of an arabinan from grape juice

An arabinan has been isolated from grape juice and purified by chromatography on polyamide and repeated ethanol precipitations. The structural identity of the arabinan was established by enzymatic degradation of the polysaccharide with a purified -L-arabinofuranosidase and methylation analysis. The results obtained suggest that the arabinan consists of an (1 → 5)-linked backbone of L-arabinofuranosyl residues to which sidechains of L-arabinose are attached in the 3-position.     

Structural features of the hemicellulose a from the stem of Mimosa bracatinga

The hemicellulose A from the stem of Mimosa bracatinga contains residues of d-xylose and 4-O-methyl-d-glucuronic acid. Smith-degradation and methylation data indicate it to consist of a backbone of (1 → 4)-linked β-d-xylopyranosyl residues with side chains consisting of single units of 4-O-methyl--d-glucosyluronic acid attached to position 3. The branched structure was also indicated by Smith-degradation and methylation analysis of the oligosaccharides obtained by enzymic hydrolysis of the hemicellulose.     

Structural studies of a mucilage from Abroma augusta root bark

The structure of an acidic polysaccharide isolated from Abroma augusta root bark was determined by sugar and methylation analyses and high resolution ¹H- and ¹³C-NMR spectroscopy. The main chain of the polysaccharide was composed of 1,2-linked - -rhamnopyranose and 1,4- or 1,3-linked - -galacturonic acid residues. The terminal β- -glucuronic acid residue was attached to the 3- and/or 4-position of the - -galacturonic acid residue.     

The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk)

The physiologically active, gel-forming fraction of the alkali-extractable polysaccharides of Plantago ovata Forsk seed husk (psyllium seed) and some derived partial hydrolysis products were studied by compositional and methylation analysis and NMR spectroscopy. Resolving the conflicting claims of previous investigators, the material was found to be a neutral arabinoxylan (arabinose 22.6%, xylose 74.6%, molar basis; only traces of other sugars). With about 35% of nonreducing terminal residues, the polysaccharide is highly branched. The data are compatible with a structure consisting of a densely substituted main chain of beta-(1-->4)-linked D-xylopyranosyl residues, some carrying single xylopyranosyl side chains at position 2, others bearing, at position 3, trisaccharide branches having the sequence L-Araf-alpha-(1-->3)-D-Xylp-beta-(1-->3)-l-Araf. The presence of this sequence is supported by methylation and NMR data, and by the isolation of the disaccharide 3-O-beta-D-xylopyranosyl-L-arabinose as a product of partial acid hydrolysis of the polysaccharide.     

Isolation and characterization of a tetrasialoganglioside from mouse brain, containing 9-O-acetyl,N-acetylneuraminic acid

A new ganglioside, containing an alkali-labile linkage, was extracted from mouse brain and purified. It represents 3.6% of total lipid-bound sialic acid in the tissue and was obtained in pure form with a yield of about 35%. It contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:1:2:1:4 and, upon exhaustive sialidase treatment gives the monosialoganglioside GM1. Partial acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry and chromium trioxide oxidation studies showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose-ceramide. Three of the four sialic acid residues are N-acetylneuraminic acid and one, as shown by gas-liquid chromatography-mass spectrometry, is 9-O-acetyl,N-acetylneuraminic acid, which contains the alkali labile linkage. 9-O-acetyl,N-acetylneuraminic acid is -ketosidically linked to position 8 of the N-acetylneuraminic acid residue bound to position 3 of the internal galactose. The other two N-acetylneuraminic acid residues form a disialosyl residue linked to position 3 of external galactose. The complete structure of the studied ganglioside is as follows: NeuAc2–8NeuAc2–3Galβ1–3GalNAcβ1–4(9-O-Ac-NeuAca2–8NeuAc2-1′-N-acylsphingosine, and it can be considered as a derivative of the tetrasialoganglioside GQ1b.     

Fractional and structural characterization of hemicelluloses isolated by alkali and alkaline peroxide from barley straw

Eight hemicellulosic fractions were obtained by sequential treatment of dewaxed barley straw with 0.1 M NaOH at 45 °C for 3 h, 0.25, 0.5, 1.0, 1.5, 2.0, and 3.0% H₂O₂ at 45 °C for 3 h at pH 11.5, and 10% KOH–1% Na₂B₄O₇·10H₂O at 28 °C for 15 h under continuous agitation. The yields of the fractions were 8.0, 3.1, 3.3, 3.3, 2.2, 2.0, 2.0, and 9.9%, respectively, of the initial amount of barley straw, corresponding to the dissolution of 21.6, 8.4, 8.9, 8.9, 5.9, 5.4, 5.4, and 26.7% of the original hemicelluloses. Meanwhile, the successive treatment also solubilized 29.1, 15.8, 14.6, 10.8, 4.5, 3.2, 2.7, and 3.7% of the original lignin, respectively. This sequential extraction together resulted in dissolution of 91.1% of the original hemicelluloses and 84.8% of the original lignin. The 0.1 M NaOH-soluble hemicellulosic fraction contained mainly xylose, glucose, and arabinose, 44.2, 15.7, and 15.2%, respectively, while the 10% KOH–1% Na₂B₄O₇·10H₂O-soluble fraction predominated in xylose, 75.0%. The six alkaline peroxide-soluble fractions were composed of 50.3–54.4% xylose, 14.7–16.9% arabinose, 6.8–10.7% glucose, 6.8–8.5% glucuronic acid or 4-O-methyl- -glucuronic acid, 0.4–1.5% mannose, and 0.3–1.2% rhamnose. All the hemicellulosic fractions contained substantial amounts of glucuronoarabinoxylans and noticeable quantities of β-glucans. In comparison, the six hemicellulosic fractions, isolated with alkaline peroxide, had much higher molecular weights (56,890–63,810 g mol⁻¹) than those of the two hemicellulosic preparations (28,000–29,080 g mol⁻¹), isolated with alkali in the absence of hydrogen peroxide. The thermal stability of the hemicelluloses increased with an increment of their molar mass.     

Characterization of a biologically active arabinogalactan from the leaves of Plantago major L.

PMIa is a Type II arabinogalactan with anti-complementary activity isolated from the leaves of Plantago major L. It has a molecular weight of 77000–80000 Da and consists of arabinose (38%), galactose (49%), rhamnose (6%), galacturonic acid (7%) and 1.5% protein with hydroxyproline, alanine and serine as the main amino acids. Characterization of PMIa by methylation and GC-MS, methanolysis and GC, Smith degradation, weak acid hydrolysis, ¹³C-NMR, ¹H-NMR, two-dimensional heteronuclear NMR and DEPT show that it consists of 1,3-linked galactan chains with 1,6-linked galactan side chains attached to position 6. The side chains are further branched in position 3 with 1,3-linked galactose residues which have 1,6-linked galactose attached to position 6; these 1,3- and 1,6-linked galactose chains altogether probably form a network. Terminal and 1,5-linked arabinose in furanose form are attached to the galactan mainly through position 3 of the 1,6-linked galactose side chains.     

The glycerol teichoic acid from the cell wall of Bacillus stearothermophilus B65

1. A glycerol teichoic acid has been extracted from cell walls of Bacillus stearothermophilus B65 and its structure examined. 2. Trichloroacetic acid-extractable teichoic acid accounted for 68% of the total cell-wall phosphorus and residual material could be hydrolysed to a mixture of products including those characteristic of glycerol teichoic acids. 3. The extracted polymer is composed of glycerol, phosphoric acid, d-glucose and d-alanine. 4. Hydrolysis of the polymer with alkali gave glycerol, 1-O-alpha-d-glucopyranosylglycerol and its monophosphates, glycerol mono- and di-phosphate, as well as traces of a glucosyldiglycerol triphosphate and a glucosylglycerol diphosphate. 5. The teichoic acid is a polymer of 18 or 19 glycerol phosphate units having alpha-d-glucopyranosyl residues attached to position 1 of 14 or 15 of the glycerol residues. 6. The glycerol residues are joined by phosphodiester linkages involving positions 2 and 3 in each glycerol. 7. d-Alanine is in ester linkage to the hydroxyl group at position 6 of approximately half of the glucose residues. 8. One in every 13 or 12 polymer molecules bears a phosphomonoester group on position 3 of a glucose residue, the possible significance of which in linkage of the polymer to other wall constituents is discussed.     

C NMR study on peracetylated derivatives of cyclomaltoheptaose (β-cyclodextrin, β-CD) and its methylated derivative

Peracetylated samples of cyclomaltoheptaose (β-cyclodextrin, β-CD) and its methylated derivative were studied by ¹³C NMR. The acetyl carbonyl carbon signal in peracetylated β-CD was resolved into a triplet, and the three peaks were assigned by long-range C---H COSY and INAPT techniques. The individual carbonyl peak was found to be indicative of the location of the acetyl group on the 2, 3, and 6 position in the glucose residues. An acetylated derivative of a partly methylated β-CD was also subjected to ¹³C NMR analysis to determine the distribution of acetyl and, subsequently, methyl groups on the glucose residues.     

Structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured Cordyceps militaris

The water-soluble crude polysaccharides were obtained from the fruiting bodies of cultured Cordyceps militaris by hot water extraction followed by ethanol precipitation. The polysaccharides were successively purified by chromatography on DEAE–cellulose-52 and Sephacryl S-100 HR columns, giving main three polysaccharide fractions termed P50-1, P70-1, and P70-2. Structural features of P70-1 were investigated by a combination of chemical and instrumental analysis, such as partial acid hydrolysis, methylation analysis, periodate oxidation – Smith degradation, GC–MS, ¹³C NMR, HPAEC-PAD, and FT-IR. The results indicated that P70-1 has a backbone of (1 → 6)-linked β-d-mannopyranosyl residues, which occasionally branches at O-3. The branches were mainly composed of (1 → 4)-linked -d-glucopyranosyl and (1 → 6)-linked β-d-galactopyranosyl residues, and terminated with β-d-galactopyranosyl residues and -d-glucopyranosyl residues. In the in vitro antioxidant assay, P70-1 was found to possess hydroxyl radical-scavenging activity with an IC₅₀ value of 0.548 mg/ml.     

Evidence of the presence of 2-O-beta-D-xylopyranosyl-alpha-l-arabinofuranose side chains in barley husk arabinoxylan

(Glucurono)arabinoxylans were extracted from barley husks and degraded with endo-beta-xylanase or subjected to periodate oxidation. The released oligosaccharide fragments were separated and isolated on Biogel-P2, and their structures were determined by NMR spectroscopy. The oligosaccharides identified consisted of beta-d-(1-->4)-linked xylopyranosyl residues, of which some were substituted at O-3 with alpha-l-arabinofuranosyl groups or at O-2 with 4-O-methylglucuronic acid. In addition to these substituents, a disaccharide side chain, 2-O-beta-d-xylopyranosyl-alpha-l-arabinofuranose, attached at position O-3 of the main chain, was proved to exist in arabinoxylan from barley husks. The compound was fully characterized with NMR, and all (1)H and (13)C NMR signals were assigned. The arabinose to xylose ratio was low (approximately 0.2) and no 2,3-disubstitution existed. No blocks of substituted xylose residues could be observed along the main chain.     

Periodate oxidation and degradation studies on the major water-soluble arabinoxylan in rye grain

The major water-soluble arabinoxylan from rye grain has previously been shown to contain a main chain of 4-linked β- -xylopyranosyl residues in which, on average, every second is substituted at position 3 with terminal - -arabinofuranosyl residues. Periodate oxidation, reduction and fragmentation by mild acid hydrolysis produced a series of glycerol xylosides containing 4-linked xylopyranosyl residues linked at the reducing end to position 2 of glycerol. It was shown that a one-step periodate oxidation was incomplete due to the formation of relatively stable hemiacetal linkages. A sequential oxidation and reduction procedure was used to bring about complete oxidation of arabinose and unbranched xylose residues in the intact polysaccharide. Quantitative analysis of the products liberated by mild acid hydrolysis revealed the presence of glycerol xylosides with one, two or three xylose residues in the molar ratio of 1·00:0·86:0·02. The xylose residues must have originated from branched residues in the main chain of the arabinoxylan. The units or small blocks of two residues are therefore distributed mainly as isolated branched residues and not randomly as previously reported.     

Isolation, characterization, and antitumor activities of the cell wall polysaccharides from Elsinoe leucospila.

A cell-wall preparation from the cells of Elsinoe leucospila, which produces elsinan extracellularly when grown on sucrose or glucose-potato extract medium, was fractionated systematically. The heteropolysaccharide that was released by treatment with Actinase E digestion, comprised D-mannose, D-galactose, and D-glucose (molar ratio, 1.5:1.0:0.1). Methylation, mild acid hydrolysis, and 13C-NMR studies suggested that the polysaccharide contains a backbone of alpha-(1----6)-linked D-mannose residues having two kinds of side chains, one attached at the O-4 with single or short beta-(1----6)-linked D-galactofuranosyl residues, and the other attached at O-2 with short side chains, most probably, of alpha-(1----3)-linked D-mannopyranosyl residues. A moderately branched D-glucan fraction, obtained from the cold alkali extract, was fractionated to give an antitumor-active purified beta-(1----3)-glucan having branches of single beta-D-glucosyl groups, one out of eight D-glucose residues being substituted at the O-6.     

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas’ disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2β protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.     

An alkali-soluble heteroxylan from seeds of Phoenix dactylifera L

Alkali-soluble polysaccharides, isolated from the seeds of dates, have been investigated using methylation and partial hydrolysis studies. The polysaccharides are shown to contain D-xylose and 4-O-methyl-D-glucuronic acid in a molar ratio of 5:1. An aldobiouronic acid from hemicellulose was characterized, and investigation revealed that the hemicellulose consists of a polymer of (1-->4)-linked D-xylopranosyl residues having branches of D-xylopyranosyl and 4-O-methyl-alpha-D-glucopyranosyluronic acid.     

Fine tuning of the spectral properties of LH2 by single amino acid residues

The peripheral light-harvesting complex, LH2, of Rhodobacter sphaeroides consists of an assembly of membrane-spanning alpha and beta polypeptides which assemble the photoactive bacteriochlorophyll and carotenoid molecules. In this study we systematically investigated bacteriochlorophyll-protein interactions and their effect on functional bacteriochlorophyll assembly by site-directed mutations of the LH2 alpha-subunit. The amino acid residues, isoleucine at position -1 and serine at position -4 were replaced by 12 and 13 other residues, respectively. All residues replacing isoleucine at position -1 supported the functional assembly of LH2. The replacement of isoleucine by glycine, glutamine or asparagine, however, produced LH2 complex with significantly altered spectral properties in comparison to LH2 WT. As indicated by resonance Raman spectroscopy extensive rearrangement of the bacteriochlorophyll-B850 macrocycle(s) took place in LH2 in which isoleucine -1 was replaced by glycine. The replacement results in disruption of the H-bond between the C3 acetyl groups and the aromatic residues +13/+14 without affecting the H-bond involving the C13(1) keto group. In contrast, nearly all amino acid replacements of serine at position -4 resulted in shifting of the bacteriochlorophyll-B850 red most absorption maximum. Interestingly, the extent of shifting closely correlated with the volume of the residue at position -4. These results illustrate that fine tuning of the spectral properties of the bacteriochlorophyll-B850 molecules depend on their packing with single amino acid residues at distinct positions.     

The structure of a polysaccharide isolated from Inonotus levis P. Karst. mushroom (Heterobasidiomycetes)

Inonotus levis biomass was extracted with 5% NaOH containing NaBH₄, the insoluble material was discarded and the solution dialyzed. It was further treated with proteinase and the polymeric fraction isolated by gel chromatography. It contained mostly a polysaccharide of the following structure:

β-GlcA-(1→2)--Gal-(1→6)--Gal3OMe-(1→[→6)--Gal-(1→6)--Gal3OMe-(1→]_5–10

where a non-reducing terminal glucuronic acid residue was present in about half of the molecules, making thus some of the chains acidic and others neutral. Methyl groups were present at about half of the galactose residues, however, no direct proof of the presence of defined repeating units was obtained due to NMR signals overlap. We believe that these short polymeric chains might be originally attached to a protein via serine or threonine residues and were cleaved off due to the alkaline conditions of extraction. Another polymer, co-extracted with this galactan, was a branched phosphorylated mannan with a structure similar to that of the mannan from Saccharomyces cerevisiae yeast.     

Synthesis of new glycyrrhetinic acid (GA) derivatives and their effects on tyrosinase activity

To synthesize glycyrrhetinic acid (GA) derivatives (3, 4, 5, 10, 13, 14, 15, and 16), we first removed the ketonic group in the C-11 position, and the carboxylic function at the C-30 position was kept intact, reduced to an alcohol, or transformed to an aldehyde corresponding derivatives 10 and 13. Glycyrrhetinic acid (GA) derivatives (3, 4, 5, 15, and 16) were coupled with 4-amino piperpyridine derivatives (12 and 14) and 4-fluorobenzyl bromide at C-30 carboxylic acid position of glycyrrhetinic acid. In subsequent tyrosinase assays, we found that GA derivatives 4, 5, and 16 were not active at early time points, but strongly inhibited tyrosinase activity at late time points. Of the GA derivatives examined, derivative 5 was most active, with an IC₅₀ value of 50 μM after 2 h reaction. IC₅₀ values of derivatives 4 and 16 were 120 and 170 μM, respectively. Further kinetic data indicated that these derivatives are slow-binding inhibitors of tyrosinase. The time-dependent inhibition was reversed when vitamin C or kojic acid was used, that is, both compounds showed active inhibition at early time points. These results suggest that GA derivatives are much more stable than vitamin C or kojic acid, although their intrinsic inhibitory potentials are relatively low. Higher stability and activity suggest that GA derivative 5 might be a useful candidate for skin whitening.