Chondroitin sulfate isolated from the secretion of the venom-producing parotoid gland of Brazilian bufonid.

The parotoid gland of bufonids is characterized as a specialized integument region, formed by different gland types. The secretion elaborated by the largest glandular alveoli has been related to animal chemical defense and is constituted by granular protein content, associated with a basophilic and alcianophilic material with features of glycoconjugates. This study aimed to identify and characterize the glycoconjugates in the secretion of the largest granular gland of the parotoid gland of Rinella icterica by histochemical and immunohistochemical techniques at light microscopy, biochemical methods, and nuclear magnetic resonance spectroscopy. Our results showed that the glycoconjugate content contains a mixture of chondroitin­6­sulfate (C6S) and chondroitin-non-sulfate (C0S). Thus, chondroitin sulfate probably plays an important role in gland physiology, probably protecting the protein content while inside the secretory portion.

Structure of the K12 capsule containing 5,7-di-N-acetylacinetaminic acid from Acinetobacter baumannii isolate D36.

The repeat unit of the K12 capsular polysaccharide isolated from the Acinetobacter baumannii global clone 1 clinical isolate, D36, was elucidated by means of chemical and spectroscopical methods. The structure was shown to contain N-acetyl-D-galactosamine (D-GalpNAc), N-acetyl-D-fucosamine and N-acetyl-L-fucosamine linked together in the main chain, with the novel sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid (5,7-di-N-acetylacinetaminic acid or Aci5Ac7Ac), attached to D-GalpNAc as a side branch. This matched the sugar composition of the K12 capsule and the genetic content of the KL12 capsule gene cluster reported previously. D-FucpNAc was predicted to be the substrate for the initiating transferase, ItrB3, with the Wzy polymerase making a α-D-FucpNAc-(1 → 3)-D-GalpNAc linkage between the repeat units. The three glycosyltransferases encoded by KL12 are all retaining glycosyltransferases and were predicted to form specific linkages between the sugars in the K12 repeat unit.

The antimutagenic effect of mistletoe lectin (Viscum album L. var. coloratum agglutinin).

A galactose- and N-acetyl-D-galactosamine-specific lectin (Viscum album L. var. coloratum agglutinin, VCA), which is known for its anticancer activity, was isolated from mistletoe. In this study, we investigated the antimutagenic potentials of VCA by using the pre-incubation method of the Ames test (Salmonella typhimurium TA98 and TA100) in the presence or absence of S9 mixture. Viscum album L. var. coloratum agglutinin was assessed for its antimutagenic properties against the mutagens 2-aminoanthracene (2AA) and furylfuramide (AF-2) for strain TA98, and sodium azide (NaN(3) ) and 2-aminoanthracene (2AA) for strain TA100. The concentrations used for this test compound were 100, 200 and 400 µg per plate. Viscum album L. var. coloratum agglutinin showed moderate, but not negligible, protective effects regarding the antimutagenic properties against the direct-acting mutagens NaN(3) and AF-2. Furthermore, VCA was more effective in preventing the mutagenicity of the indirect-acting mutagen 2-AA (in the presence of S9) when tested with both TA98 and TA100. In conclusion, this report has shown broad ranging antimutagenic effects of VCA to numerous mutagens in TA98 and TA100 Salmonella typhimurium strains. Although the data presented here cannot be applied in vivo, they can support other antimutagenic and anticarcinogenic findings for VCA.

One-pot enzymatic production of 2-acetamido-2-deoxy-D-galactose (GalNAc) from 2-acetamido-2-deoxy-D-glucose (GlcNAc).

2-Acetamido-2-deoxy-D-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-D-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.

Determination of Tn antigen released from cultured cancer cells by capillary electrophoresis.

An incomplete elongation of O-glycans in mucins has been found in epithelial cancers, leading to the expression of shorter carbohydrate structures such as Tn antigen (GalNAc-O-Ser/Thr), which has been reported to be one of the most specific human cancer-associated structures. However, there have been no appropriate physicochemical methods for the determination of Tn antigen in biological samples. In the present paper, we developed a capillary electrophoresis method for the determination of Tn antigen, and applied the method to the analysis of the expressed Tn antigen on some leukemia and epithelial cancer cells.

Identification of undecaprenyl phosphate-beta-D-galactosamine in Francisella novicida and its function in lipid A modification.

Francisella tularensis is a highly infectious pathogen that causes tularemia. Francisella lipid A contains an unusual galactosamine (GalN) unit, attached to its 1-phosphate moiety. Two genes, flmF2 and flmK, are required for the addition of GalN to Francisella lipid A, but the relevant enzymes and the GalN donor substrate have not been characterized. We now report the purification and identification of a novel minor lipid from Francisella novicida that functions as the GalN donor. On the basis of electrospray ionization mass spectrometry (ESI/MS) and NMR spectroscopy, we propose that this compound is undecaprenyl phosphate-beta-d-GalN. Approximately 0.5 mg of pure lipid was obtained from 10 g of F. novicida by chloroform/methanol extraction, followed by DEAE-cellulose chromatography, mild alkaline hydrolysis, and thin-layer chromatography. ESI/MS in the negative mode revealed a molecular ion [M - H](-) at m/z 1006.699, consistent with undecaprenyl phosphate-GalN. (31)P NMR spectroscopy showed a single phosphorus atom in the phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the sugar. (1)H NMR studies showed the presence of a polyisoprene chain and a sugar consistent with a beta-d-GalN unit. Heteronuclear multiple-quantum coherence (HMQC) analysis confirmed that nitrogen is attached to C-2 of the sugar. Purified undecaprenyl phosphate-beta-d-GalN supports the in vitro modification of lipid IV(A) by membranes of Escherichia coli cells expressing FlmK, an orthologue of E. coli ArnT, the enzyme that transfers 4-amino-4-deoxy-l-arabinose to lipid A in polymyxin-resistant strains. The discovery of undecaprenyl phosphate-beta-d-GalN suggests Francisella modifies lipid A with GalN on the periplasmic surface of the inner membrane.

Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis.

The cyst wall of Giardia intestinalis contains proteins and a novel N-acetylgalactosamine (GalNAc) polysaccharide, which is its major constituent. GalNAc is not present in growing trophozoites, but is synthesized during encystment via an inducible pathway of enzymes that produce UDP-GalNAc from fructose 6-phosphate. This report focuses on the regulation of these enzymes and thus the genes for glucosamine 6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (AGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and UDP-N-acetylglucosamine 4-epimerase (UAE) were cloned and expressed in Escherichia coli. Each of these expressed enzymes had the predicted activity and was used to generate antibodies. Northern and Western blot analyses demonstrated that both the mRNA and protein levels for all of these enzymes increase during encystment. Nuclear run-on assays of these and the previously analyzed glucosamine 6-phosphate deaminase (GNP; glucosamine 6-P isomerase) showed that all of the genes responsible for UDP-GalNAc synthesis during encystment are induced at the transcription level.

Lectin histochemical identification of carbohydrate moieties in opossum chemosensory systems during development, with special emphasis on VVA-identified subdivisions in the accessory olfactory bulb.

Lectins, sugar-binding molecules of nonimmune origin, were used in this study to describe the development of the main olfactory and vomeronasal systems in the Brazilian gray short-tailed opossum, Monodelphis domestica. A battery of seven lectins of the N-acetylgalactosamine/galactose-binding group was used. Of the seven lectins, only two, Vicia villosa agglutinin (VVA) and Griffonia (Bandeiraea) simplicifolia lectin I-isolectin B4 (GS I-B4), were specific to the vomeronasal system. The other five lectins recognized carbohydrates in both chemosensory systems, although the binding was more intense in the accessory olfactory system. Furthermore, whereas six of the lectins stained the adult opossum accessory olfactory bulb (AOB) homogeneously, the VVA lectin distinguished two regions of the AOB. Similar to the expression of olfactory marker protein (OMP) (Shnayder et al. [1993] Neuroreport 5:193-196), the rostral half of the AOB stained much darker with VVA than the caudal half, and the onset of the restricted pattern of staining at age 45 days also coincided. We conclude that 1) GS I-B4 and VVA recognize cell surface carbohydrate moieties specific to the vomeronasal, but not to the main olfactory, system, and 2) the carbohydrate moiety that is recognized by the VVA lectin, presumably terminal N-acetyl-galactosamine, is both temporally and spatially restricted in the opossum AOB. These results are discussed in the framework of other known spatially restricted molecules of the two major nasal chemosensory systems.

Structure of polygalactosamine produced by Aspergillus parasiticus.

The structure of polygalactosamine purified from the culture fluid of Aspergillus parasiticus AHU 7165 was studied. Partial acid hydrolysis of this polysaccharide, in which 55 to 65% of the monosaccharide residues were N-unsubstituted, gave a series of galactosamine oligosaccharides (dimer to hexamer) in a good yield. From the data on analyses of the polysaccharide and its oligosaccharides by gel filtration, periodate oxidation, methylation, and proton NMR measurement, the polysaccharide was characterized as a linear chain of alpha (1-4)-linked galactosamine residues. The N-unsubstituted galactosamine residues are probably distributed in a random fashion over the polysaccharide chain.

Rapid procedures for determination of endo-N-acetyl-alpha-D-galactosaminidase in Clostridium perfringens, and of the substrate specificity of exo-beta-D-galactosidases.

Culture fluid of Clostridium perfringens hydrolyzed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OPh and beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OC6H4-NO2-o or -p to beta-Gal-(1 leads to 3)-GalNAc and the aglycon. Such assays facilitated the characterization and purification of this endo-N-acetyl-alpha-D-galactosaminidase activity. This activity was purified 1200-fold by fractionation with ammonium sulfate and chromatography on columns of Sephadex-G200, DEAE-Sephadex, and hydroxylapatite. The final preparation showed activity over a broad range of pH, with an optimum at 9.0, but less-pure material had two pH optima, 4.0 and 9.0. Another assay method, which employed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-beta-GlcNAc-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 4)-beta GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, was developed for the rapid identification of the linkage specificity of exo-beta-D-galactosidases from any source via a coupled reaction with N-acetyl-beta-D-hexosaminidase.

N-Acetylgalactosamine 4,6-bissulfate in rat urine. I. Isolation, identification and chemical synthesis.

By starting with 4 1 of rat urine, it was possible to obtain a sulfate ester of hexosamine in crystalline form. A series of identification procedures including chemical analyses, enzymatic digestion, proton magnetic resonance spectroscopy and infrared spectroscopy showed that this substance is 2-acetamido-2-deoxy-D-galactose 4,6-bissulfate. The trivial name for this compound is N-acetylgalactosamine 4,6-bissulfate; Quantitation by isotopic techniques indicated the urine possessed an average concentration of 8 muM N-acetylgalactosamine 4,6-bissulfate. Further extension of these studies necessitated the chemical synthesis of N-acetylgalactosamine 4,6-bissulfate and related compounds to be used for references or as biological substrates. Direct sulfation of N-acetylgalactosamine was attempted first, and strong preference for attack on the primary hydroxyl group (position 6) was found for chlorosulfonic acid. Thus, the reaction with 2.2 molar equivalents of the sulfating agent gave N-acetylgalactosamine 6-sulfate and its derivatives bearing a second sulfate at either position 1 (minor) or position 3 (major). The lack of sulfation at position 4 could be attributed to steric effects of the sulfate group preferentially attached to position 6. Another experiment in which UDP-N-acetylgalactosamine 4-sulfate was used in place of the free sugar led to the formation of a bissulfated sugar-nucleotide which, on subsequent hydrolysis with mild acid, afforded N-acetylgalactosamine 4,6-bissulfate, the same compound as that obtained from rat urine.

The teichuronic acid of cell walls of Bacillus subtilis W23 grown in a chemostat under phosphate limitation.

Cell walls of Bacillus subtilis W23 contain teichuronic acid when grown in a chemostat under phosphate limitation at a low dilution rate, but teichoic acid at a higher dilution rate. The teichuronic acid was purified and shown to be a polymer of glucuronic acid and N-acetylgalactosamine.