Diabetic cardiomyopathy is characterized by impaired cardiac contractility leading to poor myocardial performance. We investigated the role that the hexosamine pathway, and especially altered nuclear O-GlcNAcylation, plays in the development of diabetic cardiomyopathy. Incubating neonatal rat cardiomyocytes in high glucose (25 mM) resulted in prolonged calcium transients when compared with myocytes incubated in normal glucose (5.5 mM), which is consistent with delayed myocardial relaxation. High glucose-treated myocytes also exhibited reduced sarcoendoplasmic reticulum Ca 2؉ -ATPase 2a (SERCA2a) mRNA and protein expression, decreased SERCA2a promoter activity, and increased O-GlcNAcylation of nuclear proteins compared with myocytes treated with normal glucose. Exposure of myocytes to 8 mM glucosamine or an adenovirus expressing O-GlcNAc-transferase (OGT) resulted in prolonged calcium transient decays and significantly reduced SERCA2a protein levels, whereas treatment with an adenovirus encoding O-GlcNAcase (GCA) resulted in improved calcium transients and SERCA2a protein levels in myocytes exposed to high glucose. Effects of elevated glucose or altered O-GlcNAcylation were also observed on essential transcription factors involved in cardiomyocyte function. High glucosetreated myocytes (with or without OGT adenovirus) exhibited increased levels of O-GlcNAcylated specificity protein 1 compared with control myocytes, whereas infecting high glucose-treated myocytes with GCA adenovirus reduced the degree of specificity protein 1 GlcNAcylation. Treatment of myocytes with 25 mM glucose, 8 mM glucosamine, or OGT adenovirus also significantly reduced levels of myocytes enhancer factor-2A protein compared with control myocytes, whereas infection with GCA adenovirus resulted in improved myocytes enhancer factor-2 expression. Our results suggest that the hexosamine pathway, and O-GlcNAcylation in particular, is important in impaired cardiac myocyte function and the development of diabetic cardiomyopathy.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.

SUMMARY Melanocyte stem cells in the bulge area of hair follicles are responsible for hair pigmentation, and defects in them cause hair graying. Here we describe the process of melanocyte stem cell entry into the quiescent state and show that niche-derived transforming growth factor β (TGF-β) signaling plays important roles in this process. In vitro, TGF-β not only induces reversible cell cycle arrest, but also promotes melanocyte immaturity by downregulating MITF, the master transcriptional regulator of melanocyte differentiation, and its downstream melanogenic genes. In vivo, TGF-β signaling is activated in melanocyte stem cells when they reenter the quiescent noncycling state during the hair cycle and this process requires Bcl2 for cell survival. Furthermore, targeted TGF-β type II receptor (TGFbRII) deficiency in the melanocyte lineage causes incomplete maintenance of melanocyte stem cell immaturity and results in mild hair graying. These data demonstrate that the TGF-β signaling pathway is one of the key niche factors that regulate melanocyte stem cell immaturity and quiescence.

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (NCAM), and these two polysialyltransferases often exist together in the same tissues. To determine the individual and combined roles of PST and STX in polysialic acid synthesis, in the present study we asked if PST and STX differ in the acceptor requirement and if PST and STX act together in polysialylation of NCAM. We first examined whether PST and STX differ in the requirement of sialic acid and core structures of N-glycans attached to NCAM. Polysialic acid was formed well on Lec4 and Lec13 cells, which are defective in N-acetylglucosaminyltransferase V and GDP-fucose synthesis, respectively, demonstrating that a side chain elongating from GlcNAc␤136Man␣136R and ␣-1,6-linked fucose are not required. PST and STX were found to add polysialic acid on NCAM⅐Fc molecules sialylated by ␣-2,3-or ␣-2,6-linkage in vitro, but not on NCAM⅐Fc lacking either sialic acid. These results indicate that both PST and STX have relatively broad specificity on N-glycan core structures in NCAM and no remarkable difference exists between PST and STX for the requirement of core structures and sialic acid attached to the N-glycans of NCAM. We then, using various N-glycosylation site mutants of NCAM, discovered that PST strongly prefer the sixth N-glycosylation site, which is the closest to the transmembrane domain, over the fifth site. STX slightly prefer the sixth N-glycosylation site over the fifth N-glycosylation site. The results also demonstrated that polysialic acid synthesized by PST is larger than that synthesized by STX in vitro. Moreover, a mixture of PST and STX more efficiently synthesized polysialic acid on NCAM than PST or STX alone. These results suggest that polysialylation of NCAM is influenced by the difference between PST and STX in their preference for N-glycosylation sites on NCAM. The results also suggest that PST and STX form polysialylated NCAM in a synergistic manner.Polysialic acid is a developmentally regulated carbohydrate composed of a linear homopolymer of ␣-2,8-linked sialic acid (1). NCAM 1 is highly polysialylated in embryonic tissues. In contrast, the majority of NCAM in adult tissues lacks this unique glycan, but polysialylated NCAM is present in the olfactory bulb and hippocampus of adult brain, where neural regeneration persists (2, 3). There is increasing evidence that polysialylated NCAM promotes cell migration and enhances neurite outgrowth and branching during development and neural regeneration (2-4). Polysialic acid is thought to modulate the functional properties of NCAM by rendering it less adhesive to itself (homophilic binding) (5, 6) or to other cell surface molecules (heterophilic binding). In the latter case, it has been shown that NCAM engages in interactions with L1 on the same membrane (cis-interaction) (7).The studies on NCAM knockout mice demonstrated an abnormal formation in the olfactory bulb and hippocampus and a defect in spatial learning and memory (8, 9). By using NCAM knocko...