Synthesis and biology of oligoethylene glycol linked naphthoxylosides.

Proteoglycans (PGs) are important macromolecules in mammalian cells, consisting of a core protein substituted with carbohydrate chains, known as glycosaminoglycans (GAGs). Simple xylosides carrying hydrophobic aglycons can enter cells and act as primers for GAG chain synthesis, independent of the core protein. Previously it has been shown that aromatic aglycons can be separated from the sugar residue by short linkers without affecting the GAG priming ability. To further investigate the effects of the xylose-aglycon distance on the GAG priming ability, we have synthesized xyloside derivatives with 2-naphthyl and 2-(6-hydroxynaphthyl) moieties connected to xylose, directly, via a methylene bridge, or with oligoethylene glycol linkers of three different lengths. The GAG priming ability and the antiproliferative activity of the xylosides, as well as the composition of the xyloside-primed GAG chains were investigated in a matched pair of human breast fibroblasts and human breast carcinoma cells. An increase of the xylose-aglycon distance from 0.24 to 0.37nm resulted in an increased GAG priming ability in both cell lines. Further increase of the xylose-aglycon distance did not result in any pronounced effects. We speculate that by increasing the xylose-aglycon distance, and thereby the surface area of the xyloside, to a certain level would make it more accessible for enzymes involved in the GAG synthesis. The compositions of the primed GAG chains varied with different xylosides, independent of the xylose-aglycon distance, probably due to various affinities for enzymes and/or different cellular uptake. Furthermore, no correlations between the antiproliferative activities, the xylose-aglycon distances, and the amounts or compositions of the GAG chains were detected suggesting involvement of other factors such as fine structure of the GAG chains, effects on endogenous PG synthesis, or other unknown factors for the antiproliferative activity

A Heart-Hand Syndrome Gene: Tfap2b Plays a Critical Role in the Development and Remodeling of Mouse Ductus Arteriosus and Limb Patterning

BACKGROUND: Patent ductus arteriosus (PDA) is one of the most common forms of congenital heart disease. Mutations in transcription factor TFAP2B cause Char syndrome, a human disorder characterized by PDA, facial dysmorphysm and hand anomalies. Animal research data are needed to understand the mechanisms. The aim of our study was to elucidate the pathogenesis of Char syndrome at the molecular level. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression of Tfap2b during mouse development was studied, and newborns of Tfap2b-deficient mice were examined to identify phenotypes. Gel shift assays had been carried out to search for Tfap2 downstream genes. Promoters of candidate genes were cloned into a reporter construct and used to demonstrate their regulation by Tfap2b in cell transfection. In situ hybridizations showed that the murine transcription factor Tfap2b was expressed during the entire development of mouse ductus arteriosus. Histological examination of ductus arteriosus from Tfap2b knockout mice 6 hours after birth revealed that they were not closed. Consequently, the lungs of Tfap2b(-/-) mice demonstrated progressive congestion of the pulmonary capillaries, which was postulated to result secondarily from PDA. In addition, Tfap2b was expressed in the limb buds, particularly in the posterior limb field during development. Lack of Tfap2b resulted in bilateral postaxial accessory digits. Further study indicated that expressions of bone morphogenetic protein (Bmp) genes, which are reported to be involved in the limb patterning and ductal development, were altered in limb buds of Tfap2b-deficient embryos, due to direct control of Bmp2 and Bmp4 promoter activity by Tfap2b. CONCLUSIONS/SIGNIFICANCE: Tfap2b plays important roles in the development of mouse ductus arteriosus and limb patterning. Loss of Tfap2b results in altered Bmp expression that may cause the heart-limb defects observed in Tfap2b mouse mutants and Char syndrome patients. The Tfap2b knockout mouse may add to the very limited available animal models of PDA

G-Quadruplex DNA Sequences Are Evolutionarily Conserved and Associated with Distinct Genomic Features in Saccharomyces cerevisiae

G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint

Novel aspects of vitamin C: how important is glypican-1 recycling?

The reduced form of vitamin C, ascorbic acid, is well known for its function as an antioxidant and as a protective agent against scurvy. However, many recent studies indicate other functions for vitamin C in mammalian cells. Novel findings provide possible explanations for observed beneficial effects of a high intake of vitamin C on cell growth, gene transcription, host resistance to infection, uptake of polyamines and clearance of misfolded proteins. Vitamin C exerts its effects indirectly via hypoxia-inducible factor, nitric oxide synthase and the heparan sulfate proteoglycan glypican-1, which is deglycanated in a vitamin C- and copper-dependent reaction

Involvement of Heparan Sulphate Biosynthesis and Turnover in Cell Proliferation. A Novel Role for Nitric Oxide in Recycling of Heparan Sulphate Proteoglycans.

The present investigation focuses on the role of HS metabolism in cell proliferation. The effect of the HS priming ß-D-xyloside, 2-(6-hydroxynaphthyl)-O-ß-D-xylopyranoside (Xyl-2-Nap-6-OH) on the proliferation of normal and transformed cells was studied. Xyl-2-Nap-6-OH inhibited growth of transformed cells to a significantly greater extent than normal cells. The growth inhibition exerted by the xyloside was believed to be due to its ability to prime GAG synthesis since the non-priming L-isomer did not possess any antiproliferative activity. The molecular structure of the aglycone and its ability to prime HS were other features assumed to be of importance for its antiproliferative activity. Recycling of HSPG may be a vehicle for endo/exocytosis of HS-binding growth factors and polyamines and thereby regulating cell proliferation. HSPG metabolism/recycling was therefore extensively studied in transformed endothelial cells. The major focus was on the role of NO-derived nitrite in the turnover of HSPG. As N-unsubstituted GlcN in HS chains are potential sites for NO-generated cleavage, the content and location of such residues was investigated. Transformed endothelial cells expressed a HSPG with a core protein of 60-70 kDa, which was recognised by a polyclonal antiserum raised against recombinant glypican-1 protein. In unperturbed cells, most of the radiolabelled glypican-1 carried truncated HS chains accompanied by HS oligosaccharides. Treatment with brefeldin A, which inhibits transport from the ER to the Golgi and also exit from the TGN and/or endocytosis in polarised cells, resulted in accumulation of glypican PG with full-size side chains while oligosaccharides disappeared. Treatment with suramin, an inhibitor of endoheparanase, led to partial inhibition of degradation of HS. Glypican-1 glycoforms in brefeldin A-treated cells contained long HS chains with GlcNH2 residues in multiple places, whereas unperturbed cells, suramin-treated cells, and nitrite-deprived cells contained short HS chains with only a few GlcNH2 residues. The number of GlcNH2 residues increased by combined suramin treatment and nitrite deprivation. This suggested that GlcNH2 residues and endoheparanase cleavage sites were closely located in HS chains. Pulse-chase studies clearly indicated that suramin arrested chains were the precursor of large-size PGs in the absence of de novo glypican-1 core protein synthesis. Recycling of suramin-arrested chains back to brefeldin A-arrested large PGs was precluded by nitrite deprivation. Formation of brefeldin A-arrested large glypican PGs was restored when NO-donor was supplied to nitrite-deprived cells. Taken together our data suggest a recycling of glypican-1 in transformed endothelial cells. During recycling, there is endoglycosidic cleavage of HS at or near GlcNH2 residues, and removal of these short nonreducing terminal GlcNH2 containing saccharides by NO derived nitrite would provide fresh acceptor-sites for HS chain extension. In order to assess the relationship between HS priming and antiproliferative activity of naphthol-containing ß-D-xylosides, cell proliferation assays and GAG priming studies were performed in the presence of nitrite depriving drugs. Different xylosides were synthesised and tested for growth inhibition and priming of HS synthesis. The selective growth-inhibitory effect of Xyl-2-Nap-6-OH appeared to be quite specific for this compound. Xylosides without the 6-hydroxyl, with an O-methylated 6-hydroxyl, with a free hydroxyl in a different position, or with non-fused aromatic rings were not antiproliferative. All of the tested naphthol-based xylosides were capable of priming HS synthesis. Inhibition of degradation by suramin or nitrite-deprivation resulted in increased intracellular accumulation of HS chains. Interestingly, nitrite-deprivation abrogated the growth inhibitory effect of Xyl-2-Nap-6-OH. We propose that Xyl-2-Nap-6-OH initiates synthesis of HS chains with occasional N-unsubstituted GlcN residues and that these chains can be taken up by the cells and degraded to bioactive compounds in the presence of nitrite

Isolation and Characterization of Heparan Sulfate Containing Amyloid Precursor Protein Degradation Products

Numerous studies indicate that heparan sulfate proteoglycans (HSPGs) participate in a network of complex molecular events involving amyloid precursor protein (APP) processing and formation, oligomerization, intracellular targeting, clearance, and propagation of amyloid β in Alzheimer’s disease (AD). A mutual functional interplay between recycling glypican-1 and APP processing has been demonstrated where the HS released from glypican-1 by a Cu/NO-ascorbate-dependent reaction forms a conjugate with APP degradation products and undergoes an endosome-nucleus-autophagosome co-trafficking. HS has been shown to display contradictory and dual effects in AD involving both prevention and promotion of amyloid β formation. It is therefore important to identify the source, detailed structural features as well as factors that favor formation of the neuroprotective forms of HS. Here, a method for isolation and identification of HS-containing APP degradation products has been described. The method is based on isolation of radiolabeled HS followed by identification of accompanying APP degradation products by SDS-PAGE and Western blotting