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PubMed1991

Studies on the molecular recognition of synthetic methyl beta-lactoside analogs by ricin, a cytotoxic plant lectin.

The binding of methyl beta-lactoside and of all possible monodeoxy derivatives of methyl beta-lactoside to the galactose-specific highly cytotoxin lectin ricin, has been investigated. The distribution of low-energy conformers of the disaccharide structures has been first determined using molecular-mechanics calculations and high-resolution NMR spectroscopy. The nuclear Overhauser enhancements and specific deshieldings observed are in agreement with a similar distribution of low-energy conformers for all studied compounds which may be described by a major conformer defined by phi (H1'-C1'-O1'-C4) and psi (C1'-O1'-C4-H4) torsion angles of 49 degrees and 5 degrees, respectively, with contribution of conformers with angles phi/psi 24 degrees/-59 degrees, 22 degrees/-32 degrees and 6 degrees/-44 degrees. Assuming that the disaccharides bind to the lectin in these preferred conformations, the apparent dissociation constants for the ricin-disaccharide complexes have been interpreted in terms of specific polar and nonpolar interactions. In agreement with X-ray data, the hydroxyl groups at positions 3, 4 and 6 of the beta-D-galactopyranose moiety appear as key polar groups in the interaction with ricin. These results are in contrast to previous results which have established that position 6 is not involved in lectin binding. An important nonpolar interaction involving position 3 of the beta-D-glucopyranose moiety, seems to be operative. The distribution of low-energy conformers of these disaccharide structures permits this interaction to take place with the hydroxyl group at this position intramolecularly bonded, thus rendering this region of the molecule more lipophylic in character for acceptance into nonpolar regions of the combining site.

Binding Sites↗
PubMed1997

Transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments reflect the size of the bound segment of the Forssman pentasaccharide in the binding site of Dolichos biflorus lectin.

A complex between the Forssman pentasaccharide alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D- Gal-(1-->4)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNac compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Binding Sites↗
PubMed2006

Deoxysugar transfer during chromomycin A3 biosynthesis in Streptomyces griseus subsp. griseus: new derivatives with antitumor activity.

Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-D-oliose (sugar A) and 4-O-methyl-D-oliose (sugar B), and a trisaccharide of D-olivose (sugar C), D-olivose (sugar D), and 4-O-acetyl-L-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two D-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 biosynthesis.

Antibiotics, Antineoplastic↗
PubMed1984

Mitogenic activities of synthetic lipid A analogs and suppression of mitogenicity of lipid A.

The effect of synthetic lipid A analogs on murine spleen cells was studied. The preparations represented D-glucosamine and D-glucosaminyl-beta 1,6-D-glucosamine disaccharide derivatives substituted in different combinations by ester- and amide-bound fatty acids and by phosphate groups. Significant mitogenic activity was demonstrated with a number of synthetic disaccharide preparations; however, their potency was lower than that of lipid A. The synthetic preparations were not mitogenic for spleen cells from C3H/HeJ mice. Furthermore, the mitogenicity of the synthetic preparations was abolished after binding with polymyxin B. The results indicate that for expression of mitogenicity, a phosphate group at position 1 of the reducing glucosamine and amide-bound acyloxyacyl residues are important factors. Some of the synthetic preparations containing the diglucosamine backbone and expressing relatively low mitogenicity suppressed B-cell mitogenicity of lipid A. Although these preparations were lytic for erythrocytes, they did not affect the viability of the splenic lymphocytes. Suppression was seen when the synthetic preparations were added simultaneously with or after the lipid A mitogen, but optimal suppression was expressed when the preparations were added to the system 3 h before lipid A. Washing of the cells before the addition of lipid A did not affect the results. The suppression was not due to the induction of suppressor cells by the synthetic preparations. The disaccharide preparations did not inhibit T-cell mitogenicity of concanavalin A. In contrast to the disaccharide preparations, the monosaccharide preparations suppressed mitogenicity of both lipid A and concanavalin A, probably because of their direct toxicity for lymphocytes.

Animals↗
PubMed1991

Lectinophagocytosis of encapsulated Klebsiella pneumoniae mediated by surface lectins of guinea pig alveolar macrophages and human monocyte-derived macrophages.

Macrophages express a mannose/N-acetylglucosamine-specific lectin which serves as a receptor for nonopsonic phagocytosis of mannose-coated particles. We have examined the binding to guinea pig alveolar macrophages in a serum-free medium of 16 Klebsiella pneumoniae serotypes and of the capsular polysaccharides isolated from 7 of these serotypes. Only five polysaccharides containing the repeating sequence Man alpha 2/3Man or L-Rha alpha 2/3-L-Rha bound to the macrophages. Of the 11 bacterial serotypes expressing such disaccharides in their capsular polysaccharides, 7 bound efficiently, 2 bound poorly, and 2 did not bind at all. No binding occurred with five serotypes lacking these disaccharides. Binding of the bacteria was inhibited by homologous and heterologous capsular polysaccharides that contain the disaccharide sequences, by mannan, and by (Man)25BSA (where BSA is bovine serum albumin). Man alpha 2/3Man-containing oligosaccharides were potent inhibitors compared with monosaccharides. Binding was dependent on Ca2+, modulated by cultivating the macrophages on mannan-coated surfaces, and increased in human monocyte-derived macrophages compared with monocytes. The bulk of the bacteria bound to the macrophages was internalized and killed. The data taken together suggest that Klebsiella pneumoniae cells undergo lectinophagocytosis mediated by capsular disaccharides recognized by the mannose/N-acetylglucosamine-specific lectin of macrophages. This may enhance clearance of the organisms from the serum-poor environment of the lung.

Animals↗
PubMed1996

Structural characteristics of peptidoglycan fragments required to prime mice for induction of anaphylactoid reactions by lipopolysaccharides.

Structural characteristics of peptidoglycan fragments required to prime mice for the induction of anaphylactoid reactions by Salmonella abortusequi lipopolysaccharide were examined in endotoxin-resistant C3H/HeJ mice, with special focus on the disaccharide-pentapeptide [N-acetylglucosaminyl-beta(1-4)-N-acetylmuramyl-L-alanyl-D -isoglutaminyl-meso-2,6-diaminopimelyl (DAP)-D-alanyl-D -alanine] and its smaller partial derivatives. The bacterial and synthetic muramyl tripeptides (DAP- and lysine [Lys]-type, respectively) and synthetic muramyl dipeptide primed mice for induction of anaphylactoid reactions accompanied by death within 1 h. The disaccharide-tripeptide exhibited weaker activity, and the disaccharide-tetrapeptide and muramyl tetrapeptide exhibited marginal activity. In contrast, intact peptidoglycans of various bacteria and the disaccharide-pentapeptide lacked the priming activity, although they showed adjuvant activity similar to that of the above components.

Anaphylaxis↗
PubMed2001

Synthetic 6B di-, tri-, and tetrasaccharide-protein conjugates contain pneumococcal type 6A and 6B common and 6B-specific epitopes that elicit protective antibodies in mice.

The immunogenicity and protective capacity of Streptococcus pneumoniae 6B capsular polysaccharide (PS)-derived synthetic phosphate-containing disaccharide (Rha-ribitol-P-), trisaccharide (ribitol-P-Gal-Glc-), and tetrasaccharide (Rha-ribitol-P-Gal-Glc-)-protein conjugates in rabbits and mice were studied. In rabbits, all saccharides conjugated to keyhole limpet hemocyanin (KLH) evoked high levels of pneumococcal (Pn) type 6B antibodies that facilitated type-specific phagocytosis. Unlike the disaccharide rabbit antisera, tri- and tetrasaccharide rabbit antisera also reacted with 6A PS in an enzyme-linked immunosorbent assay (ELISA) and promoted phagocytosis of 6A pneumococci. All these rabbit antisera passively protected mice against a Pn 6B challenge. The disaccharide conjugate-induced antiserum, however, failed to protect mice against a 6A challenge. In mice, phagocytic and protective anti-Pn 6B antibodies were only induced by the tetrasaccharide conjugate and not by PS 6B or PS 6B-protein conjugates. These antibodies did not cross-react with 6A PS in ELISA and were unable to phagocytize 6A pneumococci. In conclusion, the disaccharide and tetrasaccharide conjugates already contain epitopes capable of inducing 6B-specific, fully protective antibodies in rabbits and mice, respectively.

Animals↗
PubMed1980

Sporulation in Hansenula wingei is induced by nitrogen starvation in maltose-containing media.

The sexually agglutinative yeast Hansenula wingei lives in association with bark beetles that inhabit coniferous trees. This yeast was induced to sporulate by malt extract, which contains a high percentage of maltose (50%) and a low percentage of nitrogen (0.5%). A solution of 1.5% maltose without any growth factors also induced ascosporogenesis in H. wingei. Thus, only a carbon source is required for sporulation as in Saccharomyces. However, potassium acetate did not induce sporulation in H. wingei as it does in S. cerevisiae. Instead, disaccharides (such as maltose, sucrose, or cellobiose) promote sporulation better than either monosaccharides (such as dextrose, fructose, or mannose) or respiratory substrates (such as ethanol or glycerol). The specificity of disaccharides in promoting sporulation in H. wingei may be considered an adaptation since these disaccharides are present in the natural environment of this yeast. In addition, the specificity of disaccharides may be related to the induction of the disaccharidase because cells precultured on dextrose sporulate well on maltose, but cells precultured on maltose sporulate poorly on maltose. When (NH(4))(2)SO(4) was added at a low concentration (3 mM) to synthetic sporulation medium (1.5% maltose solution), sporulation was abolished, whereas other salts and nitrogen sources inhibited to a lesser extent and vitamins and trace elements had no effect. Oxygen was required for sporulation, as expected for an obligate aerobe. Maximal sporulation was achieved in 2% malt extract broth at high cell density (10(9) cells per ml), pH 5, and 25 degrees C. By using these optimal physiological conditions and hybrid strains selected from an extensive genetic breeding program, about 30% asci (10% tetrads) were obtained routinely. Thus, the genetics of cell recognition in this yeast can now be studied.

Aerobiosis↗
PubMed1985

Structure of the linkage units between ribitol teichoic acids and peptidoglycan.

The structure of the linkage regions between ribitol teichoic acids and peptidoglycan in the cell walls of Staphylococcus aureus H and 209P and Bacillus subtilis W23 and AHU 1390 was studied. Teichoic acid-linked saccharide preparations obtained from the cell walls by heating at pH 2.5 contained mannosamine and glycerol in small amounts. On mild alkali treatment, each teichoic acid-linked saccharide preparation was split into a disaccharide identified as N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol residues. The Smith degradation of reduced samples of the teichoic acid-linked saccharide preparations from S. aureus and B. subtilis gave fragments characterized as 1,2-ethylenediol phosphate-(glycerolphosphate)3-N-acetylmannosaminyl beta(1----4)N- -acetylxylosaminitol and 1,2-ethylenediolphosphate-(glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylxylosaminitol, respectively. The binding of the disaccharide unit to peptidoglycan was confirmed by the analysis of linkage-unit-bound glycopeptides obtained from NaIO4 oxidation of teichoic acid-glycopeptide complexes. Mild alkali treatment of the linkage-unit-bound glycopeptides yielded disaccharide-linked glycopeptides, which gave the disaccharide and phosphorylated glycopeptides on mild acid treatment. Thus, it is concluded that the ribitol teichoic acid chains in the cell walls of the strains of S. aureus and B. subtilis are linked to peptidoglycan through linkage units, (glycerol phosphate)3-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and (glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine, respectively.

Bacillus subtilis↗
PubMed1999

Increased sulfatation of orbital glycosaminoglycans in Graves' ophthalmopathy.

Accumulation of interstitial glycosaminoglycans (GAG) in orbital tissue of patients with Graves' ophthalmopathy (GO) leads to edema, increased orbital pressure, and proptosis. In this study, a new, highly sensitive, high performance liquid chromatography method was developed to determine the altered concentration and biochemical composition of different GAG polymers in orbital connective tissue of 27 GO patients and 18 controls. GAG were isolated by tissue homogenization and digestion, followed by sequential enzymatic GAG hydrolysis and high performance liquid chromatographic analysis of the resulting alpha,beta-unsaturated disaccharides. High recovery rates of 78 +/- 6% (mean +/- SE) and a detection limit of 4.0 microg/L (0.01 micromol/L) were obtained. Total tissue GAG amounted to 254 +/- 16 microg/g wet tissue wt in patients and 150 +/- 13 microg/g (P < 0.0001) in controls. Regarding the GAG polymers, marked differences were detected between patients and controls (chondroitin sulfate, 127 +/- 13 vs. 47 +/- 5 microg/g; hyaluronic acid, 56 +/- 5 vs. 34 +/- 4 microg/g; both P < 0.0001; dermatan sulfate, 77 +/- 6 vs. 69 +/- 6 microg/g; P < 0.05). In patients, chondroitin sulfate was the major GAG component (48 +/- 6 vs. 31 +/- 5% of total GAG in controls), whereas dermatan sulfate was dominant in controls (46 +/- 8% vs. 30 +/- 5%). The sulfated disaccharide digestion products were markedly increased (P < 0.0001) in patients, and the ratio of sulfated vs. total disaccharide content was 85 +/- 6% vs. 65 +/- 5% (P < 0.05) in patients and controls, respectively. As accumulation of negatively charged sulfate residues in GAG disaccharides results in enhanced water-binding capacity, beside inflammation and increased volume of the orbital adipose tissue, the altered structure and nature of sulfated GAG units in the orbit may be responsible for the pathogenic changes in Graves' ophthalmopathy.

Adult↗
PubMed2001

Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface.

Hyaluronan and chondroitin sulfate glycosaminoglycan secretion from retinal pigment epithelial cells was established in confluent cultures with high transepithelial resistance. Cell cultures were maintained on Millicell-PCF culture plates, which allow separation of culture medium exposed to apical and basal epithelial surfaces. Following various times in culture, apical and basal culture media were sampled at three day intervals and the glycosaminoglycan content was quantified. Samples were digested with proteinase K to free the glycosaminoglycans from their core proteins, the glycosaminoglycans were ethanol precipitated, and subjected to hyaluronidase SD and chondroitinase ABC digestion to release hyaluronan and chondroitin sulfate disaccharides. Disaccharides were fluorotagged with 2-aminoacridone, separated on polyacrylamide gels and the molar fluorescence in each disaccharide band quantitated. Hyaluronan in the apical medium was significantly higher than in the basal medium (5-12 times) at all recovery intervals (P<0.0001). In contrast, the distribution of unsulfated chondroitin, 4-sulfated chondroitin and 6-sulfated chondroitin disaccharides in apical and basal media was non-polar. Confocal microscopy of cultures probed with a hyaluronan-specific fluorotag established that the HA evident in these cultures is restricted to the apical border of the RPE cultures. Collectively, these data indicate that hyaluronan synthesized by the retinal pigment epithelium is secreted preferentially from the apical surface, suggesting that this tissue is an important source of hyaluronan present in the interphotoreceptor matrix.

Carbohydrates↗
PubMed1995

Syntheses of three interglycosidic isomers of N-acetyl-beta-D-mannosaminyl-L-rhamnoses associated with O-antigens of several gram-negative opportunistic pathogens.

We achieved practical, highly stereoselective syntheses of three interglycosidic isomers of N-acetyl-beta-D-mannosaminyl-L-rhamnoses, among which a beta(1-->4)-isomer corresponds to the repeating unit of the O-antigen of lipopolysaccharide (LPS) from the opportunistic pathogens Pseudomonas cepacia O5 and Pseudomonas aeruginosa X (Meitert). The other isomers are a beta(1-->2)-disaccharide, a constituent of LPS from Escherichia coli O1A, and an artificial beta(1-->3)-isomer. The disaccharides were obtained by simple three-step reaction sequences from 2-(benzoyloxyimino)-2-deoxyglycosyl halides (mannosamine progenitor). beta-Selective glycosylations of appropriately protected L-rhamnosyl acceptors were performed. Subsequent reduction of the 2-acyloxyimino function to an amino group, N-acetylation, and removal of the protecting groups provided the target disaccharides. 13C-NMR and nuclear Overhauser effect spectra proved to be useful for structural determination of the positional isomers of the disaccharides.

Gram-Negative Bacterial Infections↗
PubMed2001

Galactosaminoglycans from normal myometrium and leiomyoma.

In many tumors, the amount of chondroitin sulfate in the extracellular matrix has been shown to be elevated when compared to the corresponding normal tissue. Nevertheless, the degree of chondroitin sulfate increase varies widely. In order to investigate a possible correlation between the amount of chondroitin sulfate and tumor size, several individual specimens of human leiomyoma, a benign uterine tumor, were analyzed. The glycosaminoglycans from eight tumors were extracted and compared with those from the respective adjacent normal myometrium. The main glycosaminoglycan found in normal myometrium was dermatan sulfate, with small amounts of chondroitin sulfate and heparan sulfate. In leiomyoma, both dermatan sulfate and chondroitin sulfate were detected and the total amounts of the two galactosaminoglycans was increased in all tumors when compared to normal tissue. In contrast, the heparan sulfate concentration decreased in the tumor. To assess the disaccharide composition of galactosaminoglycans, these compounds were incubated with bacterial chondroitinases AC and ABC. The amounts of L-iduronic acid-containing disaccharides remained constant, whereas the concentration of D-glucuronic acid-containing disaccharides increased from 2 to 10 times in the tumor, indicating that D-glucuronic acid-containing disaccharides are responsible for the elevation in galactosaminoglycan concentration. This increase is positively correlated with tumor size.

Chondroitin Sulfates↗
PubMed1997

The utilization of multidimensional scaling to identify forage characteristics associated with preference in sheep.

Forage preference is difficult to assess but is likely an important factor in determining DM intake. In two experiments, six sheep (wethers, Exp. 1; ewes, Exp. 2) were used to obtain preference ratings on nine hays (Exp. 1) or nine fresh forages (Exp. 2). The statistical procedure of multidimensional scaling was used to develop orthogonal dimensions to account for the observed variation in preference for hays and fresh forages. This analysis produced three dimensions that accounted for 54% of the variation in mean 3-d preference for hays (Exp. 1). The three dimensions were subsequently associated by regression analysis with seven compositional variables. These were IVDMD and concentrations of monosaccharides, short-chain polysaccharides (SCP), and disaccharides in dimension 1(R2 = .99); acid detergent insoluble ash (ADIA) concentration, monosaccharide and SCP concentrations, and masticate NDF in dimension 2 (R2 = .99); and disaccharide and starch concentrations in dimension 3 (R2 = .65). Three dimensions accounting for 51% of the variation in fresh forage preference were found and related to four variables by regression analysis. These were disaccharide concentration and median particle size in dimension 1 (R2 = .76) and starch and monosaccharide + disaccharide concentrations in dimension 3 (R2 = .97). No physicochemical variables analyzed in this study were found to be significantly associated with the coordinates for the forages in dimension 2. Multidimensional scaling accounted for more than half the observed variation in animal preference and, in all but one case, these dimensions could be associated with physicochemical characteristics of the forages.

Algorithms↗
PubMed1987

Synthetic antigens as immunogens: Part II: Antibodies to synthetic T antigen.

Antibody to the carbohydrate moiety of T antigen was developed. The synthetic antigen (Gal beta 1----3 GalNAc alpha 1----OC6H4N = N-BSA) was prepared by coupling the diazonium salt of the disaccharide derivative Gal beta 1----3 GalNAc alpha 1----OC6H4NH2 (o) with bovine serum albumin. Specificity of the antibody produced was examined with structurally related synthetic saccharides using the enzyme immunoassay technique. The presence of a glycosyl group at 0-6 of either the Gal or the GalNAc residue of the disaccharide Gal beta 1----3 GalNAc did not prevent binding of the antisera to the saccharide moiety. However, the antisera did not bind either the trisaccharide moiety NeuAc2----3 Gal beta 1----3 GalNAc alpha 1----OC6H4NO2 (o) or GlcNAc beta 1----3 Gal beta 1----3 GalNAc alpha OBn. These observations indicate that antibody approach to the antigen is to the 0-3 side of the terminal galactose in the disaccharide Gal beta 1----3 GalNAc. We have also observed that the antibody prefers Gal beta 1----3 GalNAc alpha 1----to Gal beta 1----3 GalNAc beta 1----disaccharide derivatives in its binding capacity. The antibody was found to bind natural T antigen present on neuraminidase-treated red blood cells and, by immunohistochemical analysis, it was found to bind to naturally occurring T antigen on breast tumor cells.

Antibodies↗
PubMed1999

Sulphation heterogeneity in the trisaccharide (GalNAcSbeta1, 4GlcAbeta1,3GalNAcS) isolated from the non-reducing terminal of human aggrecan chondroitin sulphate.

We report here the isolation and sulphation isomer analyses of trisaccharides GalNAcS(beta1,4)GlcA(beta1,3)GalNAcS (in which S indicates sulphate) derived from the non-reducing termini of aggrecan chondroitin sulphate. Rat chondrosarcoma and human aggrecans were digested for 1 h at 37 degrees C with 30 micro-units of endo-chondroitinase ABC per microgram of chondroitin sulphate, and trisaccharides were isolated from the digests by ToyoPearl HW40S gel-filtration chromatography. Four trisaccharide species were identified; their sulphation isomer compositions, as determined by digestion with chondroitinase ACII and fluorescence-based ion-exchange HPLC, were GalNAc4Sbeta1,4GlcAbeta1,3GalNAc4S, GalNAc4Sbeta1,4GlcAbeta1,3GalNAc6S, GalNAc4,6Sbeta1,4GlcAbeta1, 3GalNAc4S and GalNAc4,6Sbeta1,4GlcAbeta1,3GalNAc6S. The abundances of such sequences in chondroitin sulphate on aggrecan from normal (foetal to 72 years of age) and from osteoarthritic human knee cartilages were also established. The results showed that non-reducing terminal GalNAc4S or GalNAc4,6S can be linked to either a 4-sulphated or a 6-sulphated disaccharide, suggesting that the sulphation of the last disaccharide might not have a direct effect on the specificity of chondroitin sulphate terminal GalNAc sulphotransferases. Furthermore, for each aggrecan preparation examined, the 4S-to-6S ratio of all chain interior disaccharides was equivalent to that in the last repeating disaccharides at the non-reducing terminus, suggesting that neither chondroitin 4-sulphotransferase nor chondroitin 6-sulphotransferase shows preferential activity near the chain terminus.

Adolescent↗
PubMedDate not supplied

Determination of residual moisture in lyophilized protein pharmaceuticals using a rapid and non-invasive method: near infrared spectroscopy.

This study examined the application of near-infrared (NIR) spectroscopy to analyze residual moisture in lyophilized protein pharmaceuticals sealed in glass vials. We demonstrated that NIR was able to determine residual moisture in five marketed and clinical products with the same precision as Karl Fischer titration. We further investigated how changes in product configuration and protein formulation affected NIR measurement accuracy using a lyophilized monoclonal antibody rhuMAb E25 containing 1% to 5% residual moisture. The results indicated that the lyophilized cake porosity and dimensions had no effect on NIR measurement when the cake height and diameter exceeded the NIR penetration depth. In addition, changing the buffer and surfactant concentrations in the formulation did not affect moisture determination by NIR. However, doubling or halving the concentration of a disaccharide, which was used as a lyoprotectant, caused significant deviation between the NIR and Karl Fischer data because the NIR absorbance of the disaccharide overlapped with the moisture signal. Furthermore, complete removal of the disaccharide resulted in alteration of the protein NIR spectra, suggesting that NIR may be used to evaluate solid-state protein structure. The disaccharide concentration must be kept constant in this formulation to obtain accurate moisture results by NIR.

Calibration↗
PubMed1992

Nutritional intervention in acute diarrhea: is a lactose-free formula essential?

A prospective study was done to determine the incidence of disaccharide intolerance among 3-36 month-old patients with acute watery diarrhea who were on breast feeding and/or lactose-containing formula. The effect of feeding intervention on the outcome was investigated. Significant disaccharide intolerance was defined as one with (1) biochemical derangements: stool pH < 6.0 (Riedel de Haen pH paper) and reducing substances > or = 0.5 mg% (Clinitest) on two consecutive determinations and (2) clinical evidence: high purging rate (> 10 gm/kg/hour) and reappearance of dehydration and/or weight loss while on a lactose containing milk. Seven of 92 patients (7.8%) had biochemical evidence of disaccharide malabsorption on admission. Subsequent monitoring of the study population showed absence of disaccharide intolerance. Despite a lactose containing formula, a significant (p < 0.05) reduction in stool output from the first to the second day (145.85 +/- 130.26 vs 115.43 +/- 95.65 g/kg admission weight) was noted. Likewise, weight gain from admission to discharge (4.56 +/- 3.44%) was observed. The mean total duration of illness (4.75 +/- 2.84 days) was well within the usual course of five to seven days. This study supports the current recommendation of continued breast feeding and/or use of lactose containing formula during acute watery diarrhea.

Acute Disease↗