Inhibition of CD44 N- and O-linked glycosylation decreases endometrial cell lines attachment to peritoneal mesothelial cells.

The attachment of endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) to peritoneal mesothelial cells (PMCs) with and without inhibition of N- and O-linked glycosylation, the viability of EECs and ESCs, and the expression of CD44 surface density were evaluated. Inhibition of CD44 N- and O-linked glycosylation by using tunicamycin and/or B-GalNAc statistically significantly inhibited endometrial cell attachment to peritoneal mesothelial cells, suggesting a role in establishment of early endometriotic lesions.

Predicting biological functions of compounds based on chemical-chemical interactions.

Given a compound, how can we effectively predict its biological function? It is a fundamentally important problem because the information thus obtained may benefit the understanding of many basic biological processes and provide useful clues for drug design. In this study, based on the information of chemical-chemical interactions, a novel method was developed that can be used to identify which of the following eleven metabolic pathway classes a query compound may be involved with: (1) Carbohydrate Metabolism, (2) Energy Metabolism, (3) Lipid Metabolism, (4) Nucleotide Metabolism, (5) Amino Acid Metabolism, (6) Metabolism of Other Amino Acids, (7) Glycan Biosynthesis and Metabolism, (8) Metabolism of Cofactors and Vitamins, (9) Metabolism of Terpenoids and Polyketides, (10) Biosynthesis of Other Secondary Metabolites, (11) Xenobiotics Biodegradation and Metabolism. It was observed that the overall success rate obtained by the method via the 5-fold cross-validation test on a benchmark dataset consisting of 3,137 compounds was 77.97%, which is much higher than 10.45%, the corresponding success rate obtained by the random guesses. Besides, to deal with the situation that some compounds may be involved with more than one metabolic pathway class, the method presented here is featured by the capacity able to provide a series of potential metabolic pathway classes ranked according to the descending order of their likelihood for each of the query compounds concerned. Furthermore, our method was also applied to predict 5,549 compounds whose metabolic pathway classes are unknown. Interestingly, the results thus obtained are quite consistent with the deductions from the reports by other investigators. It is anticipated that, with the continuous increase of the chemical-chemical interaction data, the current method will be further enhanced in its power and accuracy, so as to become a useful complementary vehicle in annotating uncharacterized compounds for their biological functions.

Distribution and reuse of 76Se-selenosugar in selenium-deficient rats.

Nutritional selenium compounds are transformed to the common intermediate selenide and then utilized for selenoprotein synthesis or excreted in urine mostly as 1beta-methylseleno-N-acetyl-Dd-galactosamine (selenosugar). Since the biological significance of selenosugar formation is unknown, we investigated their role in the formation of selenoenzymes in selenium deficiency. Rats were depleted of endogenous natural abundance selenium with a single stable isotope ((82)Se) and then made Se-deficient. (76)Se-Selenosugar was administered intravenously to the rats and their urine, serum, liver, kidneys and testes were subjected to speciation analysis with HPLC inductively coupled argon plasma mass spectrometry. Most (76)Se was recovered in its intact form (approximately 80% of dose) in urine within 1 h. Speciation analysis revealed that residual endogenous natural abundance selenium estimated by (77)Se and (78)Se was negligible and distinct distributions of the labeled (76)Se were detected in the body fluids and organs without interference from the endogenous natural abundance stable isotope. Namely, intact (76)Se-selenosugar was distributed to organs after the injection, and (76)Se was used for selenoprotein synthesis. Oxidation to methylseleninic acid and/or hydrolysis of the selenoacetal group to methylselenol were proposed to the transformation of selenosugar for the reuse. Effective use of an enriched stable isotope as an absolute label in hosts depleted of natural abundance isotopes was discussed for application in tracer experiments.

Mouse N-acetylgalactosamine 4-sulfotransferases-1 and -2. Molecular cloning, expression, chromosomal mapping and detection of their activity with GalNAcbeta1-4GlcNAcbeta1-octyl.

N-Acetylgalactosamine 4-sulfotransferase (GalNAc4ST) transfers sulfate to position 4 of nonreducing terminal GalNAc residues. We previously cloned human GalNAc4ST-1 cDNA. In this paper, we report the cloning, characterization and chromosomal mapping of mouse GalNAc4ST-1 and GalNAc4ST-2. Mouse GalNAc4ST-1 and GalNAc4ST-2 contain single open reading frames that predict type II transmembrane proteins composed of 417 and 413 amino acid residues, respectively. The amino acid sequence identity between the two isoforms is 49%. When the cDNA was transfected to COS-7 cells, sulfotransferase activities toward carbonic anhydrase VI and GalNAcbeta1-4GlcNAcbeta1-octyl were overexpressed, but the sulfotransferase activity toward chondroitin showed no increase over the control level. Northern blot analysis showed that the 2.4 kb messages of GalNAc4ST-1 and GalNAc4ST-2 were strongly expressed in the kidney, where both of the human isoforms were hardly expressed. Reverse transcription-PCR analysis showed that, unlike human GalNAc4ST-1, the expression of mouse GalNAc4ST-1 in the pituitary gland was only marginal, while that of GalNAc4ST-2 in the pituitary gland was as high as that in the kidney. These results suggest that the functions of the two GalNAc4ST isoforms may differ between human and mouse. By fluorescence in situ hybridization, the GalNAc4ST-1 and GalNAc4ST-2 genes were localized to mouse chromosome 7B3 distal-B5 proximal and chromosome 18A2 distal-B1 proximal, respectively.

Selenosugars are key and urinary metabolites for selenium excretion within the required to low-toxic range.

Essential micronutrient selenium is excreted into the urine andor expired after being transformed to methylated metabolites. Monomethylated selenium is excreted into the urine in response to a supply within the required to low-toxic range, whereas tri- and dimethylated selenium increase with excessive supply at a toxic dose. Here we show that the major urinary selenium metabolite within the required to low-toxic range is a selenosugar. The structure of 1beta-methylseleno-N-acetyl-d-galactosamine was deduced from the spectroscopic data and confirmed by chemical synthesis. This metabolite was also detected in the liver, and an additional metabolite increased with inhibition of methylation. The latter metabolite was again a selenosugar conjugated with glutathione instead of a methyl group and was assumed to be a precursor for methylation to the former metabolite. A metabolic pathway for the urinary excretion of selenium, i.e., from the glutathione-S-conjugated selenosugar to the methylated one, was proposed. Urinary monomethylated (selenosugar) and trimethylated selenium can be used as specific indices that increase within the required to low-toxic range and with a distinct toxic dose, respectively.

Alveolar macrophages bind and phagocytose allergen-containing pollen starch granules via C-type lectin and integrin receptors: implications for airway inflammatory disease.

Recent studies suggest that IgE-independent mechanisms of airway inflammation contribute significantly to the pathophysiology of allergic airway inflammatory diseases such as asthma. Such mechanisms may involve direct interactions between inhaled allergens and cells of the respiratory tract such as macrophages, dendritic cells, and epithelial cells. In this study, we investigated receptor-mediated interactions occurring between alveolar macrophages and allergen-containing pollen starch granules (PSG). We report here that PSG are released from a range of grass species and are rapidly bound and phagocytosed by alveolar macrophages. Human monocyte-derived dendritic cells also bound PSG but no internalization was observed. Phagocytosis of PSG was dependent on Mg2+ and Ca2+ and was inhibited by neo-glycoproteins such as galactose-BSA and N-acetylgalactose-BSA. Partial inhibition of phagocytosis was also seen with the Arg-Gly-Asp-Ser (RGDS) motif and with an anti-CD18 mAb (OX42). The combination of both neo-glycoprotein and anti-CD18 achieved the greatest degree of inhibition (>90%). Together, these data suggest a role for both C-type lectins and beta2-integrins in the binding and internalization of PSG. The consequences of this interaction included a rapid up-regulation of inducible NO synthase mRNA and subsequent release of NO by alveolar macrophages. Thus, receptor-mediated recognition of inhaled allergenic particles by alveolar macrophages may represent a potential mechanism for modulating the inflammatory response associated with allergic airway diseases such as asthma.

Synthesis of alpha-C(1-->3)-mannopyranoside of N-acetylgalactosamine, a new beta-galactosidase inhibitor.

Radical C-glycosidation of a 3-methylidene-7-oxabicyclo[2.2.1]heptan-2-one derivative with acetobromomannose gave a alpha-C-mannopyranoside that was converted into alpha-D-ManpCH2(1-->3)-D-GalNAc, a C-disaccharide that inhibits beta-galactosidase from jack bean with IC50 = 9.4 microM and Ki = 7.5 microM (mixed mode of inhibition).

Structural features of the combining site region of Erythrina corallodendron lectin: role of tryptophan 135.

The role of Trp 135 and Tyr 108 in the combining site of Erythrina corallodendron lectin (ECorL) was investigated by physicochemical characterization of mutants obtained by site-directed mutagenesis, hemagglutination-inhibition studies, and molecular modeling, including dynamics simulations. The findings demonstrate that Trp 135 in ECorL: (1) is required for the tight binding of Ca2+ and Mn2+ to the lectin because mutation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) contributes to the high affinity of methyl alpha-N-dansylgalactosaminide (MealphaGalNDns) to the lectin; and (3) is solely responsible for the fluorescence energy transfer between the aromatic residues of the lectin and the dansyl group in the ECorL-MealphaGalNDns complex. Docking of MealphaGalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as required for efficient fluorescence energy transfer, in one of the two possible conformations that this ligand assumes in the bound state. In the W135A mutant, which still binds MealphaGalNDns strongly, the dansyl group may partially insert itself into the place formerly occupied by Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assumes an open conformation. However, a small fraction of the W135A molecules must be able to bind MealphaGalNDns in order to explain the relatively high affinity, as compared to galactose, still remaining for this ligand. A model for the molecular events leading to inactivation of the W135A mutant upon demetallization is also presented in which the cis-trans isomerization of the Ala 88-Asp 89 peptide bond, observed in high-temperature dynamics simulations, appears not to be a required step.

Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid.

When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.