Preferential binding of E. coli with type 1 fimbria to D-mannobiose with the Manα1 → 2Man structure.

Manα1 → 2Man, Manα1 → 3Man, Manα1 → 4Man, and Manα1 → 6Man were converted to the glycosylamine derivatives. Then, they were mixed with monobenzyl succinic acid to obtain their amide derivatives. After removing the benzyl group by hydrogenation, the succinylamide derivatives were coupled with the hydrazino groups on BlotGlyco™ beads in the presence of water-soluble carbodiimide. d-Mannobiose-linked beads were incubated with fluorescence-labeled Escherichia coli with type 1 fimbria, and the number of the fluorescent dots associated with the beads was counted in order to determine the binding preference among d-mannobiose isomers. The results showed that the bacteria bind strongly to Manα1 → 2Man1 → beads, Manα1 → 3Man1 → beads, Manα1 → 4Man1 → beads, and Manα1 → 6Man1 → beads, in order. In the presence of 0.1 M methyl α-d-mannopyranoside, most of the bacteria failed to bind to these beads. These results indicate that E. coli with type 1 fimbria binds to all types of d-mannobiose isomers but preferentially to Manα1 → 2Man disaccharide.

Enhanced expression of the ß4-N-acetylgalactosaminyltransferase 4 gene impairs tumor growth of human breast cancer cells.

Two ß4-N-acetylgalactosaminyltransferases (ß4GalNAcTs), ß4GalNAcT3 and ß4GalNAcT4, have been shown to be involved in the synthesis of the GalNAcß1 â†’ 4GlcNAc (LacdiNAc) group expressed on the outer branches of N- and/or O-glycans, and only ß4GalNAcT4 is expressed in human mammary gland. We found that the expression level of the LacdiNAc group decreases as human breast cancers progress. To investigate biological significances of this disaccharide in human breast cancers, we transfected the FLAG-tagged ß4GalNAcT4 cDNA into MDA-MB-231 cells, and obtained several clones showing enhanced expression of the gene. Clones 1 and 2 showed 15 and 9 times more transcript than mock-transfected cells. The FLAG-ß4GalNAcT4 protein and its product, the LacdiNAc group, were detected in clone 1 and 2 cells. No change was observed in their growth rates while significant decreases in colony forming and invasive abilities were observed for clone 1 and 2 cells. When clone 1 cells were transplanted subcutaneously into nude mice, no tumors were formed while tumors were formed with mock-transfected cells. These results indicate that the expression of the LacdiNAc group is quite important for the suppression of malignancies of the MDA-MB-231 cells.

Gene expression levels of ß4-galactosyltransferase 5 correlate with the tumorigenic potentials of B16-F10 mouse melanoma cells.

Our previous studies showed that mouse ß4-galactosyltransferase 5 (ß4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the ß4GalT5 gene. We isolated a stable clone named E5 whose ß4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous ß4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the ß4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by ß4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.

Comprehensive analysis of endoplasmic reticulum-enriched fraction in root tips of soybean under flooding stress using proteomics techniques.

Flooding is a serious problem for soybean cultivation because it markedly reduces growth and grain yields. Here, 2 proteomics techniques were used to evaluate whether endoplasmic reticulum (ER)-enriched fraction is altered in soybean under flooding stress. Two-day-old soybeans were treated with flooding for 2 days, and rough ER-enriched fraction was then purified from root tips. Flooding-responsive protein of ER-enriched fraction was identified using gel-free and 1D-gel based proteomics techniques, and 117 proteins were increased and 212 proteins were decreased in soybean root tips in response to flooding stress. Among the identified proteins, 111 were functionally categorized as being involved in protein synthesis, post-translational modification, protein folding, protein degradation, and protein activation. Among differentially regulated proteins, the mRNA expression levels of 14 proteins that were predicted to be localized in the ER were analyzed. Notably, 3-ketoacyl-CoA reductase 1 was up-regulated and eight genes related to stress, hormone metabolism, cell wall and DNA repair were down-regulated within 1 day under flooding conditions. In addition, the expression of luminal-binding protein 5 was specifically induced in flood-stressed roots, whereas arabinogalactan protein 2 and methyltransferase PMT2 were down-regulated. Taken together, these results suggest that flooding mainly affects the function of protein synthesis and glycosylation in the ER in root tips of soybean.