Chemical synthesis of homogeneous erythropoietins proved that their bioactivity in vivo is regulated by the glycosylation pattern.

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This paper describes the study of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration from 3'-phosphoadenosine-5'-phosphate (PAP) for use in practical syntheses of carbohydrate sulfates which are catalyzed by sulfotransferases. Among the regeneration systems, the one with recombinant aryl sulfotransferase proved to be the most practical. This regeneration system was coupled with a sulfotransferase-catalyzed reaction, using a recombinant Nod factor sulfotransferase, for the synthesis of various oligosaccharide sulfates that were further glycosylated using glycosyltransferases.

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm β-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.

[reaction: see text] The ring flip of a carbohydrate is employed for the tongs-like movable component of a metal ion sensor. A pair of separated pyrene groups attached to the carbohydrate component are placed side by side when it recognizes metal ions, affording excimer fluorescence. This novel molecular sensor is selective for Zn(2+) and Cd(2+).

Objective-Tumor necrosis factor (TNF)-␣-induced endothelial injury, which is associated with atherosclerosis, is mediated by intracellular reactive oxygen species. Iron is essential for the amplification of oxidative stress. We tested whether TNF-␣ accelerated iron accumulation in vascular endothelium, favoring synthesis of hydroxyl radical. Methods and Results-Diverse iron transporters, including iron import proteins (transferrin receptor [TfR] and divalent metal transporter 1 [DMT1]) and an iron export protein (ferroportin 1 [FP1]) coexist in human umbilical endothelial cells (HUVECs). TNF-␣ caused upregulation of TfR and DMT1 and downregulation of FP1, which were demonstrated in mRNA as well as protein levels. These changes in iron transporters were accompanied by accumulation of iron that was both transferrin-dependent and transferrin-independent. Modifications of these mRNAs were regulated posttranscriptionally, and were coordinated with activation of binding activity of iron regulatory protein 1 to the iron responsive element on transporter mRNAs. Using a salicylate trap method, we observed that only simultaneous exposure of endothelial cells to iron and TNF-␣ accelerated hydroxyl radical production. Conclusions-TNF-␣ could cause intracellular iron sequestration, which may participate importantly in the pathophysiology of atherosclerosis and cardiovascular disease. Key Words: cytokines Ⅲ endothelium Ⅲ free radicals Ⅲ inflammation Ⅲ iron A therosclerosis now is generally recognized as a chronic inflammatory condition, and inflammatory cytokines such as tumor necrosis factor (TNF)-␣ have been associated with the development of atherosclerotic lesions and consequent cardiovascular events. 1,2 Dysfunction and loss of vascular endothelial cells, which provide a nonthrombogenic surface and a permeability barrier, occur early in atherosclerosis. 3 Several lines of evidence has suggested that TNF-␣-induced cell injury is mediated through its ability to promote intracellular reactive oxygen species (ROS) formation. 4,5 Among these species, superoxide anion (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ) are not very reactive, and usually are neutralized by an elaborate antioxidant defense system. However, transition metal-catalyzed Haber-Weiss reaction can transform O 2 Ϫ and H 2 O 2 to hydroxyl radical, which are extremely powerful oxidizing species. 6,7 Iron is an essential element required for biochemical reactions subserving a wide variety of functions in cells and organisms. 8 However, free iron, possibly the most important transition metal in biologic systems, can act as an electron donor. 6,9 Excessive intracellular accumulation of iron therefore could amplify the damaging effect of oxidative stress in inflammatory conditions, leading to cell injury.The recent identification of iron transport proteins has rapidly expanded our knowledge of molecular aspects of iron processing, especially in the reticuloendothelial system and small intestine. 10,11 Such proteins include transferrin receptor (TfR), divalent metal tr...

Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.

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