Lignin-Carbohydrate Complexes Containing Phenolic Acids Isolated from the Culms of Bamboo

Lignin-carbohydrate complexes containing phenolic acids (LCC-W) were isolated from Moso-bamboo (Phyllostachys pubescens Mazel) and characterized. LCC-W was separated into three fractions (W-l, 2, and 3) by gel filtration on Sepharose 4B. Of these three LCCs W-2 and W-3 were included in the gel matrices. W-2 consists of 34.7% neutral sugar, 1.6% uronic acid, 52.1% lignin, and 6.1% phenolic acid, and W-3, 67.9%, 3.6%, 22.3%, and 1.1%, respectively. Neutral sugar residues of W-2 and W-3 were mainly l-arabinose and d-xylose in the ratios of 5.5:94.3 in W-2, and 4.5:95.0 in W-3, respectively. Methylation, periodate degradation, and NMR analyses indicated that the carbohydrate moiety of LCC-W is composed of a linear backbone of β-(l→4)-linked d-xylopyranose residues with approximately every thirty residues carrying one 4-O-methyl-d-glucuronic acid and one or two arabinofuranose residues. Saponified phenolic acids were composed of trans-p-coumaric and trans-ferulic acids, which seems to be esterified to carbohydrate and lignin, respectively. Alkaline treatment, periodate degradation, and hydrophobic interaction chromatography suggested the presence of alkali labile and stable lignin–carbohydrate linkages.     

Water-Soluble Glycoconjugates of Vegetables I. Isolation and Properties of Arabinogalactan-Proteins from Cabbage

We present a new method for isolating and purifying water-soluble arabinogalactan-proteins from cabbage and give their chemical properties. The water-soluble nondialyzable material from fresh cabbage was separated into three fractions (A-I, II, and III) by gel filtration on Sepharose CL-4B. A-I and A-II can be purified by HPLC. Borate is necessary to avoid formation of insoluble aggregates during isolation and purification. The molecular weights of A-I, II, and III were estimated to be 4.0×10⁵, 1.0×10⁵, and 1.0~4.0×10⁴, respectively. A-I and A-II are arabinogalactan-proteins with different carbohydrate/protein ratios: 5.5/1 for A-I and 11.4/1 for A-II. The carbohydrate moieties of A-I and A-II were both arabino-3,6-galactans having d-galactose/l-arabinose ratios of 1.9/1 and 1.5/1, respectively. The amino acid composition indicates an abundance of hydroxyproline, serine, threonine and alanine, the sum of which amounted to about 50% of the total amino acids. A-I contained 1.5 times more hydroxyproline (20%) than A-II (14%), while A-II contained higher proportions of serine, glycine, and alanine. A-III was not a glycoprotein but was a mixture of carbohydrate and polypeptides.     

Bibliography

Water-soluble polysaccharides isolated from the roots of Panax ginseng C. A. Meyer were completely fractionated into two neutral fractions (WGPN and WGPA-N) and six acidic fractions (WGPA-1-RG, WGPA-2-RG, WGPA-1-HG, WGPA-2-HG, WGPA-3-HG and WGPA-4-HG) by a combination of ethanol precipitation, ion-exchange and gel permeation chromatographies. The analytical results showed that WGPN was a starch-like glucan; WGPA-N was a mixture of starch-like glucan and arabinogalactan; WGPA-1-RG and WGPA-2-RG were composed of major neutral sugars and minor acidic sugars that belong to the type-I rhamnogalacturonan (RG-I)-rich pectins, while fractions WGPA-1-HG to WGPA-4-HG were mainly composed of galacturonic acid (GalA, 62.4–92.1%) and have been identified to be homogalacturonan (HG)-rich pectins with different degrees of methyl-esterification, ranging from 0% to 30%. High performance gel permeation chromatography (HPGPC) showed that the six acidic fractions were homogenous, with molecular weights approximately ranging from 3.5 × 10³ to 1.1 × 10⁵. Lymphocyte proliferation assays showed that both the neutral polysaccharides and acidic polysaccharides were potent B and T cell stimulators.     

Decomposition of [14C]Lignocelluloses of Spartina alterniflora and a Comparison with Field Experiments

Decomposition of lignocelluloses from Spartina alterniflora in salt-marsh sediments was measured by using ¹⁴C-labeled compounds. Rates of decomposition were fastest in the first 4 days of incubation and declined later. Lignins labeled in side chains were mineralized slightly faster than uniformly labeled lignins; 12% of the [side chain-¹⁴C]lignin-labeled lignocellulose was mineralized after 816 h of incubation, whereas only 8% of the [U-¹⁴C]lignin-labeled lignocelluloses were degraded during this period. The carbohydrate moiety within the lignocellulose complex was degraded about four times faster than the lignin moiety; after 816 h of incubation, 29 to 37% of the carbohydrate moiety had been mineralized. Changes in concentration of lignin and cellulose in litter of S. alterniflora were followed over 2 years of decay. Cellulose disappeared from litter more rapidly than lignin; 50% of the initial content of cellulose was lost after 130 days, whereas lignin required 330 to 380 days for 50% loss. The slow loss of lignin compared with other litter components resulted in a progressive enrichment of litter in lignin content. The rates of mineralization of [¹⁴C]lignocelluloses in marsh sediments were similar to the rates of lignocellulose decomposition in litter on the marsh.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected.We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80°C for 1 min, and purified by ultrafiltration and ethanol precipitation.The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem. Biophys. Res. Commun. 63, 875–880).A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB³H₄. The polysaccharide was only slightly degraded (2%) by jack bean α-mannosidase and could precipitate 15 times its own weight of concanavalin A.The acidic polymer was also detected in the cell “periplasm” and was secreted from cells grown in defined media during the period of decelerating growth.     

Studies on pig serum lipoproteins. IV. Isolation and characterization of glycopeptides from pig serum low density lipoprotein.

Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.     

Differences between the carbohydrate units of cell-surface glycoproteins of moust B- and T-lymphocytes.

Carbohydrate units of cell-surface glycoproteins of mouse B- and T-lymphocytes, labelled in their sialic acid residues by the periodate/NaB3H4 method and in their galactose residues by the galactose oxidase/NaB3H4 method after neuraminidase treatment, have been studied. Glycopeptides were prepared from the labelled cells by Pronase digestion and fractionated by concanavalin A affinity chromatography into two fractions (A and B). Alkali-labile oligosaccharides were isolated after mild NaOH/NaBH4 treatment by gel filtration. The alkali-labile oligosaccharides were further analysed by t.l.c. To study the relative proportion of neutral mannose-rich carbohydrate units (fraction C) in lymphocyte glycoproteins, glycopeptides were also prepared from unlabelled cells and subjected to concanavalin A affinity chromatography after N-[3H]acetylation of their peptide moiety. The major alkali-labile oligosaccharide component of both cell types was identified as galactosyl-(beta 1 leads to 3)-N-acetylgalactosaminitol. T-Lymphocytes were characterized by a high proportion of this oligosaccharide and a lower proportion of alkali-stable fraction A glycopeptides, whereas the opposite was observed for B-lymphocytes. The relative proportions of the concanavalin A-binding fractions B and C were similar in both cell types. The differences observed may correlate with the different surface properties of B- and T-lymphocytes.     

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: I. Isolation and some properties of the glycoproteins.

An acidic heteropolysaccharide preparation derived from the mycelium of Fusarium sp. M7-1 was fractionated into two fractions, precipitable and nonprecipitable, by treatment with cetyltrimethylammonium bromide (Cetavlon). These two fractions were further purified to apparent homogeneity on ultracentrifugation by treatment with charcoal and gel filtration chromatographies. Two glycoproteins, precipitable GP I and nonprecipitable GP II, were obtained. The molecular weights of GP I and GP II were estimated to be about 8.8 x 10(4) and 3.7 x 10(4), respectively, on gel filtration chromatography. Both GP I and GP II contained a high proportion of serine and threonine. Treatment of GP I and GP II with alkaline solution resulted in an increase in absorbance at 240 nm. Alkaline borohydride treatment markedly decreased the number of seryl and threonyl residues and resulted in an increase in alanine and the formation of 2-aminobutyric acid. It also resulted in release of low and high molecular weight carbohydrate chains. From these results, we conclude that both GP I and GP II are glycoproteins with carbohydrate chains attached to the protein moiety through O-glycosidic linkages to the hydroxyl group of serine and/or threonine.     

Polysaccharides of diatoms occurring in Lake Baikal

Polysaccharide composition of neutral, acid- and alkali-soluble fractions of the diatoms Stephanodiscus meyerii Genkal et Popovsk and Aulacoseira baicalensis (K. Meyer) Simonsen of Lake Baikal has been studied. Neutral polysaccharides were represented by chrysolaminarans (1-->3;1-->6-beta-D-glucans). The chrysolaminaran from S. meyerii consists of the high- and low-molecular-weight fractions (40 and 2-5 kDa, respectively) and contains a large number of beta-1-->6-bound glucose residues. The chrysolaminaran from A. baicalensis is a low-molecular-weight 1-->3:1-->6-beta-D-glucan containing a small number of beta-1-->6 bonds, with mannitol being attached to the reducing unit of its chain. Acid- and alkali-soluble polysaccharide fractions are practically absent in S. meyerii. The alkali-soluble fraction from A. baicalensis is a low-molecular-weight (2-kDa) glycoprotein, the carbohydrate moiety of which is represented by a heteropolysaccharide.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes.

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1----4GlcNAc beta 1----3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.     

Preparation of 3H-labeled serum glycoproteins from antarctic fish

Radioactive glycoproteins were prepared from the purified serum glycoproteins of antarctic fish by introducing tritium atoms (³H) into their carbohydrate moiety. The carbohydrate moiety is composed of disaccharides, β-D-galactosyl-(1→4) α-N-acetylgalactosamine, which accounts for 60% of the molecule. The hydroxyl groups (OH) at the carbon-6 positions of both sugar residues were oxidized with galactose oxidase to aldehyde groups and subsequently reduced to hydroxyl groups (O³H) with sodium borohydride-³H (NaB³H₄). Under suitable conditions, the end products (tritiated glycoproteins) are highly radioactive and retain their original unique freezing point depressing activity.     

Studies on bael (Aegle marmelos) seed glycoproteins

The carbohydrate material isolated from bael (Aegle marmelos) seeds was resolved into four, pure glycoprotein fractions. The carbohydrate moiety of one of the fractions (F-I) contained galactose, glucose, arabinose and rhamnose in the molar ratios of 6:2:8:3. The linkages among these monosaccharide residues and the anomeric configurations of the glycosyl residues were determined. The structure at the glycosyl-amino acid junction was also established.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus.

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the period of decelerating growth.     

Isolation and Structural Characterization of Lignin Polymer from Dendrocalamus sinicus

Dendrocalamus sinicus, which is the largest bamboo species in the world, has broad prospects in the fields of bioenergy and biorefinery application. In this study, dewaxed D. sinicus samples were sequentially treated with 80 % ethanol containing 0.025 M HCl, 80 % ethanol containing 0.5 % NaOH, and aqueous alkaline solutions (containing 2.0, 5.0, and 8.0 % NaOH, respectively) at 75 °C for 4 h, in which 9.63, 8.71, 21.83, 21.09, and 13.09 % of the original lignin were isolated, respectively. The lignin fractions obtained were comparatively characterized by chemical composition, molecular weights, and structural features by wet chemical and instrumental analysis methods. It was found that the bamboo lignin fractions isolated by ethanol had lower weight-average molecular weights (1,360–1,380 g?mol^?1) and contained much higher amounts of associated hemicelluloses, while the lignin fractions isolated by aqueous alkaline solutions had higher weight-average molecular weights (5,300–6,040 g?mol^?1) and contained lower amounts of associated hemicelluloses. Spectroscopy analyses indicated that the bamboo lignin was a typical grass lignin, consisting of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. A small percentage of the lignin side-chain was found to be acetylated at the γ-carbon, predominantly at syringyl units. The major interunit linkages present in the bamboo lignin obtained were β-O-4′ aryl ether linkages, together with lower amounts of β-β′, β-5′, and β-1′ linkages.     

Impact of residue quality on the C and N mineralization of leaf and root residues of three agroforestry species

A laboratory incubation experiment with ¹⁵N labeled root and leaf residues of 3 agroforestry species (Leucaena leucocephala, Dactyladenia barteri and Flemingia macrophylla) was conducted under controlled conditions (25 C) for 56 days to quantify residue C and N mineralization and its relationship with residue quality.No uniform relation was found between the chemical composition of the above and below residues. The leucaena and dactyladenia roots contained more lignin (8 and 26% respectively) and less N (2.0 and 1.0% respectively) than the respective leaves (2 and 13% lignin and 2.9 and 1.4% N, respectively), whereas the differences between the lignin and N contents of the flemingia leaves and roots were not significant (4.6 and 3.0% lignin and 2.63 and 2.68% N, respectively). The leucaena leaves contained more polyphenols than the roots (6.4 and 3.6%), while the polyphenol content of the leaves and roots of the other residues was similar (5.0 and 5.1% for dactyladenia and 4.0 and 3.5% for flemingia).Three patterns of N mineralization could be distinguished. A first pattern, followed by residues producing the highest amounts of CO₂, showed an initial immobilization of soil derived N, followed by a net release of both soil and residue derived N after 7 days of incubation. A second pattern, followed by the flemingia leaf residues which produced intermediate amounts of CO₂ and had an intermediate quality, showed no significant immobilization of soil derived N, and significant mineralization of residue N. A third pattern, followed by both low quality dactyladenia residues, showed a low release of residue derived N and a continued inmobilization of soil derived N.Residue C mineralization was significantly (p<0.05) correlated with the residue lignin content, C-to-N ratio, and polyphenol-to-N ratio. The proportion of residue N mineralized (immobilized) after 56 days of incubation was significantly correlated with the residue N content (p<0.01) and the C-to-N ratio (p<0.05). The relations were quadratic, rather than linear. The ratio of the proportion of residue N mineralized (immobilized) over the proportion of residue C mineralized after 56 days was highly significantly correlated with the lignin content (p<0.01) and C-to-N (p<0.001), lignin-to-N (p<0.01), polyphenol-to-N (p<0.01) and (lignin+polyphenol)-to-N ratios (p<0.01) in a linear way. This indicates that due to the low availability of the residue C, relatively less N is immobilized for the very low quality residues ((lignin+polyphenol)-to-N ratio: 29.7) than for the residues with a relatively higher quality ((lignin+polyphenol)-to-N ratios between 3.3 and 12.5).     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Fractionation and characterization of polysaccharides from abaca fibre

Abaca fibre polysaccharides were fractionated into water soluble, pectic, 1% NaOH soluble, hemicellulosic and cellulose fractions by extraction with hot water, dilute hydrochloric acid (pH 1.6), aqueous 1% NaOH and 17.5% NaOH, respectively. Cellulose (60.4–63.6%) and hemicelluloses (20.8%) were the major polysaccharides in abaca fibres. The hot water soluble polysaccharides contained noticeable amounts of pectic substances and a large proportion of neutral polysaccharides. The pectic polysaccharide preparation was enriched in both galacturonic acid and neutral sugars, including xylose, glucose, galactose, arabinose, and rhamnose. Extraction of the fibre with aqueous 1% NaOH produced the hemicellulose–lignin complex, which was enriched in xylose and, to a lesser extent, glucose-, arabinose- and galactose-containing polysaccharides, together with 7.6% associated lignin. Further extraction of the delignified fibre residue with aqueous 17.5%. NaOH removed the hemicellulose fractions, which were strongly enriched in xylose-containing polysaccharides. Besides ferulic and p-coumaric acids, six other phenolic monomers were also detected in the mixtures of alkaline nitrobenzene oxidation of associated lignin in all the polysaccharide fractions. The content of bound lignin in water soluble, pectic, and 1% NaOH soluble polysaccharides (Fractions 1, 2, and 3), isolated directly from the lignified fibres, was 12 times that of the hemicellulosic preparations (Fractions 4 and 5) isolated from the delignified fibre residues.     

Levels of aflatoxin B1, bacteria and fungi in feed and food in 1971 — 1987

Totally 39% out of 8371 feed and their component samples were contaminated by aflatoxin B₁. Mean contamination was 36μg/kg with maximum yield 10100 μg/kg. Contamination of samples by total count of organisms, mean contamination and maximum yield, respectively was: 1) bacteria 99%, 2.2×10⁶, 2.4×10⁸; 2) proteolytic bacteria 94%, 1.2×10⁵, 3.0×10⁶;3) moulds 98%, 1.3×10⁵, 9.0×10⁶; 4) yeasts 44 %, 3.3×10⁴, 3.6×10⁶. The samples were contaminated in 92 % byAspergillus spp, in 71% byAspergillus flavus, in 83% byPenicillium spp, and in 20% byFusarium spp with mean contamination 8.3×104, 1.1×10³, 4.2×10⁴, 5.0×10³ , and maximum yield 6.8×10⁶, 1.0×10⁵, 5.0×10⁶, 1.5×10⁶, respectively. Totally 8.5% of strains were aflatoxinogenic and 4.4% of the strains were isolated from feed and 21 % of the strains from grain/nut.     

Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation,liberation, and fractionation of oligosaccharides and reaction with lectins

Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc, dGalβ1 → 3dGlcNAcβ1 → 3dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, dGalβ1 → 4dGlcNAcβ1 → d 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with ³H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB³H₄ at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.