Preparative use of the analytical column of a sugar autoanalyzer for resolution of gluco-oligosaccharides of the same molecular weight.

A sugar autoanalyzer was used on a preparative scale to resolve a gluco-oligosaccharide mixture. In this way the components of the following mixtures were resolved: O-alpha-D-glucopyranosyl-(1-3)-O-[alpha-D-glucopyranosyl-(1-6)]-D-glucose (1), O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-3)-D-glucose (2) and O-alpha-D-glucopyranosyl-(1-3)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (3), O-alpha-D-glucopyranosyl-(1-3)-O-alpha-D-glucopyranosyl-(1-4)-D-glucose (4) and O-alpha-D-glucopyranosyl-(1-4)-O-alpha-D-glucopyranosyl-(1-3)-D-glucose (5), and O-alpha-D-glucopyranosyl-(1-2)-O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (6) and O-alpha-D-glucopyranosyl-(1-3)--O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (7).     

Structural analysis of leuconostoc dextrans containing 3-O-α-d-glucosylated α-d-glucosyl residues in both linear-chain and branch-point positions,or only in branch-point positions,by methylation and by 13C-N.M.R. spectroscopy

It had been established by methylation-structural analysis that dextran fraction S from Leuconostoc mesenteroides NRRL B-1355 has two types of α-d-glucopyranosyl residues that are linked through O-3, i.e., 35% of the residues carry a (1→3)-bond, and ～10% carry a (1→6)-bond in addition to a (1→3)-bond. Two similarly constituted dextrans have now been identified by methylation-structural analysis, namely, the S-type fractions from L. mesenteroides strains NRRL B-1498 and B-1501. The S-type fractions from L. mesenteroides strains B-1355, B-1498, and B-1501 are structurally differentiated from the α-d-glucans (characteristically insoluble) of certain cariogenic Streptococci which also contain both 3-O- and 3,6-di-O-substituted α-d-glucopyranosyl residues. ¹³C-N.m.r. spectra have been recorded at 90° for both the S- and L-type fractions of strains B-1355, b-1498, and B-1501. The L-type fractions have a low degree of branching through 3,6-di-O-substituted αd-glucopyranosyl residues, but no 3-mono-O-substituted residues. (Dextran fraction S of Streptococcus 5000 g.l.c. instrument equipped with hydrogen-flame detectors. On-column injection of glass columns (2 mm i.d. x 1.23 m) was employed for all such chromatography.The ¹³C-n.m.r. conditions and methods for preparation of dextran samples have been described(su4). In general, a Varian XL-100-15 spectrometer equipped with a Nicolet TT-100 system was employed in the Fourier-transform mode. Chemical shifts are expressed in p.p.m. relative to external tetramethylsilane, but were actually calculated by reference to the lock signal.     

IgA anti-dextran B1355 responses.

Injection of mice bearing the Ig-1a allotype with dextran B1355 results in an IgM antibody response that is generally regarded as thymus independent. Moreover, the antibody is directed to alpha[1,3] determinants on dextran B1355 and shares cross-reacting idiotypic determinants with a lambda 1 IgA (J558) myeloma protein as well as a lambda 1 IgM (MOPC 104E) myeloma protein. In this study, we show that BALB/c (Ig-1a) mice injected with dextran B1355 produced highly significant IgA anti-dextran responses with specificity directed to the alpha[1,3] epitope. Kinetics of the IgA anti-dextran response in BALB/c mice paralleled kinetics of the IgM response. However, the magnitude of the IgA response was markedly T cell dependent and age dependent.     

Estimation of antibodies specific for dextran.

Methods are described for the isolation and characterization of picogram quantities of anti-dextran antibodies. 14C-dextrans produced by using the dextransucrases of Leuconostoc mesenteroides strains B1355 and B512 were used in a radioimmunoassay. The specificity of this assay was verified by using cell cytoplasmic lysates from mouse plasmacytomas, J558 (anti-alpha 1 leads to 3 dextran) and W3129 (anti-alpha 1 leads to 6 dextran). Dextran produced by strain B1355 and insolubilized with epichlorohydrin was used as an immunoabsorbent.     

Some structural features of an insoluble α-D-glucan from a mutant strain of Leuconostoc mesenteroides NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces two soluble extracellular α-D-glucans from sucrose: alternan and dextran. An unusual mutant strain derived from NRRL B-1355 has recently been isolated which produces practically no soluble polysaccharide, but significant amounts of an insoluble D-glucan. Methylation analysis shows it contains linear (1→3) and (1→6) linkages as well as (1→2) and (1→3) branch linkages. The insoluble glucan was partially digestible by endodextranase, giving rise to a series of oligosaccharides, a high-molecular weight soluble fraction and an insoluble residue. Treatment of the soluble dextranase-limit fraction with an α(1→2) debranching enzyme led to further dextranase susceptibility. Methylation, FTIR and NMR analyses of the dextranase-treated fractions indicate a non-uniform structure with domains bearing similarities to L. mesenteroides strain NRRL B-1299 dextran and to insoluble streptococcal D-glucans. Received 05 November 1998/ Accepted in revised form 31 March 1999     

Genes on different chromosomes influence the antibody response to bacterial antigens

B6.C congenic strains of mice, possessing histocompatibility (H) alleles from high responding BALB/cBy (C) mice on the genetic background of low responding C57BL/6By (B6) mice, were assayed for their ability to make an antibody response to Type III pneumococcal polysaccharide (SSS-III) and the (13) epitope of bacterial (Leuconostoc) dextran B-1355. The results affirmed that the antibody response to SSS-III is multigenic and that genes making a positive contribution to responsiveness are located on different chromosomes, i. e., chromosomes 1, 3, 4, 5, and 9. At least one other gene also influences responsiveness to SSS-III; it is linked to the H-17 locus, which has not yet been assigned to a specific chromosome. Genes on chromosomes 1, 4, and 5 influence the magnitude of the antibody response to dextran B-1355. Some of these genes may be antigen-specific in their mode of action; however, others may not since they appear to exert a positive influence on the antibody response to both SSS-III and dextran B-1355.     

A comparison of the IgG isotype expression in the so-called thymus-independent immune responses to alpha(1----3) and alpha(1----6) dextran

This paper presents data on the IgG antibody response against two "thymus-independent" dextran (Dex) antigens from Leuconostoc mesenteroides, alpha(1----3) Dex B 1355S and alpha(1----6) Dex B 512F in BALB/c and C57BL/6 mice, which are considered to be responders or low responders to the respective antigen. The data point toward three common rules governing the two anti-Dex responses despite immunogenetic and antigenic disparities: (1) age dependency of the IgG isotype regulation of the response; (2) down-regulation of IgG isotype expression by T cells; and (3) individually determined preposition for IgG isotype formation in a given animal.     

Specific and non-specific affinities of the extracellular glucosyltransferase complex of Streptococcus mutans 6715

Glucosyltransferases (GTF) from different strains of streptococci exhibited different elution profiles when fractionated on insoluble-dextran affinity columns. The proportions of unadsorbed and adsorbed GTF were not related to their extent of stimulation by exogenous dextran, and GTF preparations exposed to, and freed from, clinical dextran prior to fractionation lost their ability to bind to the dextran columns. Different proportions of bound GTF were released by irrigation of columns with different concentrations of salt and clinical dextran, and the “specific” binding and release of GTF exhibited by a column possessing covalently linked, clinical dextran ligands was duplicated on a control column that did not possess the dextran ligands. These results, and the high affinity of GTF for hydrophobic alkyl (Shaltiel) ligands, demonstrate that ionic and hydrophobic properties of impure GTF aggregates may lead to erroneous characterization of the dextran affinity of some protein fractions. Fractionations on DEAE-Sepharose and on hydroxylapatite showed that the two dextran-dependant GTF activities (GTF-S and GTF-I) were present in the major enzyme fraction (Streptococcus mutans 6715) recovered from a Sephacryl S-200 affinity column. A minor, dextran-independent GTF was not adsorbed onto the Sephacryl column. The presence of SDS (0.005%) and Triton X100 (0.01%) stabilized GTF activity during gel filtration and improved the separation of GTF-S and GTF-I in hydroxylapatite fractionation of the highly aggregated enzyme. A comparable separation of the two enzyme forms on DEAE-Sepharose was achieved only if T10 dextran (10 mg/mL) was included with the detergent mixture in the column irrigant.     

Regulation of clones responding to dextran B1355S. III. The thymic-independent and thymic-dependent antibody responses

The primary antibody response of different mouse strains challenged with two antigenic forms of alpha (1-3) dextran, dextran B1355S and dextran-hemocyanin, was examined. Only BALB/c mice responded with both kappa and lambda antibodies. The kappa to lambda ratio was affected by factors such as the antigenic form of dextran, the time at which the serum was analyzed, and the priming regimen. Surprisingly, priming with hemocyanin in adjuvant increased the kappa portion of the response not only to dextran-hemocyanin but also to dextran B1355S. Other strains of mice responded with only lambda antibodies. These results extend our previous results on the analysis of dextran-specific B cell precursors.     

Three glycoproteins with antimutagenic activity identified in Lactobacillus plantarum KLAB21

Antimutagenic substances were purified from a culture supernatant of Lactobacillus plantarum KLAB21 cells isolated from kimchi, a Korean traditional fermented vegetable, and their characteristics were investigated. The antimutagenic substances were separated into two fractions by DEAE-cellulose ion-exchange column chromatography, which were designated the R1 and R2 fractions. The R1 fraction was then divided into two fractions again by Sephadex G200 gel filtration chromatography, and the fractions were designated R1-1 and R1-2. All three fractions were further purified using a Sepharose CL-6B gel filtration column. All the purified fractions were successfully stained with fuchsin as well as Coomassie brilliant blue, suggesting that they are glycoproteins. The purified fractions were confirmed to possess antimutagenic activity against N-methyl-N'-nitro-N-nitrosoguanidine on Salmonella enterica serovar Typhimurium TA100 cells. Their molecular masses were determined to be 16 (R1-1), 11 (R1-2), and 14 (R2) kDa on the Sepharose CL-6B column. Total sugar contents were 8.4% (R1-1), 7.3% (R1-2), and 9.4% (R2). The amino acid compositions of the fractions were different from each other; the major amino acids were glutamic acid (21.5%) and phenylalanine (17.1%) in the R1-1 fraction and glycine (41.3%) in the R1-2 fraction, but valine (31%) and phenylalanine (22.6%) were the major amino acids in the R2 fraction.     

High-temperature enhancement of 13C-N.M.R. chemical-shifts of unusual dextrans,and correlation with methylation structural analysis

Dextran fractions from NRRL strains Leuconostoc mesenteroides B-742, B-1299, B-1355, and Streptobacterium dextranicum B-1254 were examined by ¹³C-n.m.r. spectroscopy at 34 and 90°, and by methylation structural analysis. The native, structurally homogeneous dextran from L. mesenteroides NRRL B-1402 was also examined. The data allow correlations to be made between the structure and physical properties of the S (soluble) and L (less-soluble) fraction pairs of dextrans B-742, B-1254, B-1299, and B-1355. For the dextrans under consideration here, increasing solubility of the dextran (both in water and in aqueous ethanol) was found to correlate with decreasing percentages of α-d-(1→6)-linked d-glucopyranosyl residues. Both the diagnostic nature of the 70–75-p.p.m. spectral region with regard to type of dextran branching, and the increase in resolution of the polysaccharide spectra at higher temperatures, have been further confirmed.     

Immunochemical studies on dextrans: distribltion of alpha1 leads to 2 and alpha1 leads to 6 specific determinants of dextran NRRL B1397.

A rabbit anti-dextran serum was separated into two fractions, specific for α1→2 and α1→6 glucose linkages, respectively. Dextran NRRL B1397 containing α1→2 and α1→6 specific determinants was fractionated by step-wise precipitation with the α1→2 specific antibody fraction to see whether the α1→2 and α1→2 specific determinants were both in the same molecule. Most of the dextran fractions were shown to have two specificities by quantitative inhibition experiments with kojibiose and isomaltose as inhibitors. The precipitin lines in agar of dextran fractions with antibody fractions specific to α1→2 and α1→6 fused. These results strongly suggest that these two antigenic determinants are present in the same molecule.     

Insoluble Glucan Formation by Leuconostoc mesenteroides B-1355

Leuconostoc mesenteroides B-1355 produced at least three glucosyltransferases (GTFs). We previously identified GTF-2 as alternansucrase and GTF-3 as fraction L dextransucrase. We here show that GTF-1 is a previously unreported sucrase that synthesized water-insoluble dextran. Our evidence consisted of the following. (i) GTF-1 was a major component and GTF-2 was a minor component of culture supernatant fractions, but supernatant fractions actively synthesized water-insoluble glucan. (ii) GTF-1 and culture supernatants produced an unusual high-pressure liquid chromatography pattern of malto-oligosaccharides that was not reproduced by GTF-2-GTF-3 mixtures. (iii) GTF-2, GTF-3, and GTF-2-GTF-3 mixtures did not synthesize insoluble glucan from sucrose. Nearly all of the alternansucrase in young (less than 17-h) cultures was associated with the cells.     

Three Glycoproteins with Antimutagenic Activity Identified in Lactobacillus plantarum KLAB21

Antimutagenic substances were purified from a culture supernatant of Lactobacillus plantarum KLAB21 cells isolated from kimchi, a Korean traditional fermented vegetable, and their characteristics were investigated. The antimutagenic substances were separated into two fractions by DEAE-cellulose ion-exchange column chromatography, which were designated the R1 and R2 fractions. The R1 fraction was then divided into two fractions again by Sephadex G200 gel filtration chromatography, and the fractions were designated R1-1 and R1-2. All three fractions were further purified using a Sepharose CL-6B gel filtration column. All the purified fractions were successfully stained with fuchsin as well as Coomassie brilliant blue, suggesting that they are glycoproteins. The purified fractions were confirmed to possess antimutagenic activity against N-methyl-N′-nitro-N-nitrosoguanidine on Salmonella enterica serovar Typhimurium TA100 cells. Their molecular masses were determined to be 16 (R1-1), 11 (R1-2), and 14 (R2) kDa on the Sepharose CL-6B column. Total sugar contents were 8.4% (R1-1), 7.3% (R1-2), and 9.4% (R2). The amino acid compositions of the fractions were different from each other; the major amino acids were glutamic acid (21.5%) and phenylalanine (17.1%) in the R1-1 fraction and glycine (41.3%) in the R1-2 fraction, but valine (31%) and phenylalanine (22.6%) were the major amino acids in the R2 fraction.     

Regulation of clones responding to α 1 → 3 dextran: I. Individual variation in the expression of idiotypes

Balb/c mice were immunized with dextran B1355S and assayed for serum antibodies and direct plaque-forming cells. The earliest detectable anti-dextran response occurred in 2-week-old animals. Anti-idiotypic antisera against MOPC-104E and J558 were raised in A/He mice and rendered individual idiotype specific by cross-absorption. When the amounts of MOPC-104E and J558 idiotypes in immune sera and the PFC response of individual mice were analyzed, the ratio of both idiotypes were found highly variable in all tested animals. This observed individual variability in the expression of two major idiotypes in the Balb/c response to dextran B1355S provides an experimental basis for investigating the mechanisms regulating idiotype expression.     

Inspection of active sites of human salivary alpha-amylase isozymes by means of non-reducing-end substituted maltooligosaccharides with 2-pyridylamino residue

The modes of action of four alpha-amylase isozymes, which were purified from human saliva, on p-nitrophenyl alpha-maltopentaoside (G5P), maltohexaitol (G6R), and their 2-pyridylamino derivatives, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG5P) and O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O- alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D- glucitol (FG6R) were examined at various pH values. No differences in their modes of action on the substrates was found. Irrespective of which enzyme was used, the molar ratio of the hydrolysis products of G5P or G6R was almost constant at any pH examined. On the other hand, those of FG5P and FG6R varied with pH such that predominantly O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG3) was formed at high pH ranges, while the formation of O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-gl ucose (FG4) increased at lower pH. The result indicates that the binding mode of FG5P or FG6R to the active sites of the enzymes changed with pH; namely, interactions between the 2-pyridylamino residue of the substrates and some amino acid residue(s) located in the active sites were influenced by pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: Specificities and reactivities of the antibodies formed in rabbits to stearyl-isomaltosyl oligosaccharides

The specificities and the sizes and shapes of the antibody combining sites of the 15 antisera raised against various stearyl-isomaltosyl oligosaccharides were studied by quantitative precipitin and precipitin inhibition. The antibodies precipitated well with dextrans B512 and B1424 but less well with B1299S and B1355S. Only 3 of the 15 antisera reacted with linear dextrans; however, with about 50% of the added antibodies being precipitated, showing that most of the antibodies cannot bind to internal determinants along the dextran molecules and are similar to myeloma protein W3129 in having cavity-type sites which bind only to terminal nonreducing ends of α1 → 6 dextran. Antibodies differing in the sizes of their antibody combining sites were elicited in different rabbits by the same antigen. Of the 15 antisera studied, four have antibody combining sites as large as IM3, five as large as IM4, three as large as IM5 and three as large as IM6. The association constants for various isomaltose oligosaccharides of an antiserum (R-862) showing fewest bands in isoelectric focusing gel were determined by affinity electrophoresis and were comparable to W3129.     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal anti-alpha (1----3) dextran antibodies of Igha BALB/c and Ighb C.B20 mice display striking similarities

Allotype Ighb congenic C.B20 mice when immunized with dextran B1355S are unable to produce anti-alpha (1----3) dextran antibodies that express the VH-associated cross-reactive IdX idiotype. This intrastrain-specific idiotype is normally associated only with the anti-dextran response of Igha mice of which BALB/c is a prototype strain. In this study we have obtained monoclonal hybridoma antibodies specific for the alpha (1----3) glucosidic linkage of dextran from C.B20 mice that were presensitized with rabbit anti-IdX antibodies. These antibodies display the light chain isotype distribution, the H chain amino terminal sequence, share VH-associated IdX idiotypic determinants, and finally the similar fine specificity for dextrans observed for anti-alpha (1----3) dextran antibodies of BALB/c mice.     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis.

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)