Functional network in posttranslational modifications: Glyco-Net in Glycoconjugate Data Bank.

Elucidating pathways related to posttranslational modifications (PTMs) such as glycosylation is of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids, and various biological events, are considered extremely valuable and convenient tools for the systematic investigation of PTMs. Glyco-Net (http://bibi.sci.hokudai.ac.jp/functions/) can dynamically make network figures among various biological molecules and biological events. A certain molecule or event is expressed with a node, and the relationship between the molecule and the event is indicated by arrows in the network figures. In this chapter, we mention the features and current status of the Glyco-Net and a simple example of the search with the Glyco-Net.

Hyperadiponectinemia enhances bone formation in mice.

BACKGROUND: There is growing evidence that adiponectin, a physiologically active polypeptide secreted by adipocytes, controls not only adipose tissue but also bone metabolism. However, a role for adiponectin in bone development remains controversial. METHODS: We therefore investigated the endocrine effects of adiponectin on bone metabolism using 12-week-old male transgenic (Ad-Tg) mice with significant hyperadiponectinemia overexpressing human full-length adiponectin in the liver. RESULTS: In Ad-Tg mice, the serum level of osteocalcin was significantly increased, but the levels of RANKL, osteoprotegerin, and TRAP5b were not. Bone mass was significantly greater in Ad-Tg mice with increased bone formation. In contrast, bone resorption parameters including the number of osteoclasts and eroded surface area did not differ between Ad-Tg and their littermates. CONCLUSIONS: These findings demonstrate that hyperadiponectinemia enhances bone formation in mice.

Multicentric extra-abdominal desmoid tumors arising in bilateral lower limbs.

Extra-abdominal desmoid tumors preferentially affect the shoulders, arms, backs, buttocks, and thighs of young adults. Multicentric occurrence is rather rare but seems to be another distinctive feature of extra-abdominal desmoid tumors. In this article we report a rare case of multicentric extra-abdominal desmoid tumors arising in bilateral lower limbs.

Isolation and characterization of a novel peptide, osteoblast activating peptide (OBAP), associated with osteoblast differentiation and bone formation.

A long-standing goal in bone loss treatment has been to develop bone-rebuilding anabolic agents that can potentially be used to treat bone-related disorders. To purify and isolate a novel anabolic that acts to osteoblasts, we monitored changes in intracellular calcium concentrations ([Ca(2+)]i). We identified a novel, 24 amino-acid peptide from the rat stomach and termed this peptide osteoblast activating peptide (OBAP). Furthermore, we examined the effects of OBAP in osteoblasts. First, osteoblast differentiation markers (alkaline phosphatase [ALP], osteocalcin [OCN]) were analyzed using quantitative RT-PCR. We also examined the ALP activity in osteoblasts induced by OBAP. OBAP significantly increased the expression of osteoblast differentiation markers and the activity of ALP in vitro. Next, to address the in vivo effects of OBAP on bone metabolism, we examined the bone mineral density (BMD) of gastrectomized (Gx) rats and found that OBAP significantly increased BMD in vivo. Finally, to confirm the in vivo effects of OBAP on bone, we measured serum ALP and OCN in Gx rats and found that OBAP significantly increased serum ALP and OCN. Taken together, these results indicate that the novel peptide, OBAP, positively regulates bone formation by augmenting osteoblast differentiation. Furthermore, these results may provide a new therapeutic approach to anabolically treat bone-related disorders.

N-myc downstream-regulated gene 1/Cap43 expression promotes cell differentiation of human osteosarcoma cells.

The N-myc downstream regulated gene 1 (NDRG1)/Cap43 is closely associated with cell differentiation, and its expression is induced by hypoxia and increasing intracellular calcium levels. Whether the NDRG1/Cap43 expression in cancer cells is a predictive marker of good or poor prognosis in patients, depends upon tumor types and differentiation status. In this study, we examined whether the NDRG1/Cap43 expression was involved in the differentiation of osteosarcoma cells, using three osteosarcoma cell lines, MG63, U2OS and SaOS2. The NDRG1/Cap43 expression in MG63 and U2OS was significantly enhanced by vitamin D3, which also induced the production of osteocalcin, a differentiation marker of osteoblasts. The knockdown of NDRG1/Cap43 using small interfering RNA also suppressed the production of osteocalcin and enhanced cell proliferation, accompanied by the suppression of p21 expression. Furthermore, the acquired invasiveness of osteosarcoma cells during the invasion in Matrigel resulted in the decreased expression of NDRG1/Cap43. On the basis of these results, our proposed role for NDRG1/Cap43 would be in the capacity of differentiation and invasion in osteosarcoma cells.

Functional network of glycan-related molecules: glyco-net in glycoconjugate data bank.

BACKGROUND: Glycans are involved in a wide range of biological process, and they play an essential role in functions such as cell differentiation, cell adhesion, pathogen-host recognition, toxin-receptor interactions, signal transduction, cancer metastasis, and immune responses. Elucidating pathways related to post-translational modifications (PTMs) such as glycosylation are of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids and various biological events are considered extremely valuable and convenient tools for the systematic investigation of PTMs. However, there is no database which dynamically draws functional networks related to glycans. DESCRIPTION: We have created a database called Glyco-Net http://www.glycoconjugate.jp/functions/, with many binary relationships among glycan-related molecules. Using search results, we can dynamically draw figures of the functional relationships among these components with nodes and arrows. A certain molecule or event corresponds to a node in the network figures, and the relationship between the molecule and the event are indicated by arrows. Since all components are treated equally, an arrow is also a node. CONCLUSIONS: In this paper, we describe our new database, Glyco-Net, which is the first database to dynamically show networks of the functional profiles of glycan related molecules. The graphical networks will assist in the understanding of the role of the PTMs. In addition, since various kinds of bio-objects such as genes, proteins, and inhibitors are equally treated in Glyco-Net, we can obtain a large amount of information on the PTMs.

Structure of pleiotrophin- and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches.

Endogenous pleiotrophin and hepatocyte growth factor (HGF) mediate the neurite outgrowth-promoting activity of chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains isolated from embryonic pig brain. CS/DS hybrid chains isolated from shark skin have a different disaccharide composition, but also display these activities. In this study, pleiotrophin- and HGF-binding domains in shark skin CS/DS were investigated. A high affinity CS/DS fraction was isolated using a pleiotrophin-immobilized column. It showed marked neurite outgrowth-promoting activity and strong inhibitory activity against the binding of pleiotrophin to immobilized CS/DS chains from embryonic pig brain. The inhibitory activity was abolished by chondroitinase ABC or B, and partially reduced by chondroitinase AC-I. A pentasulfated hexasaccharide with a novel structure was isolated from the chondroitinase AC-I digest using pleiotrophin affinity and anion exchange chromatographies. It displayed a potent inhibitory effect on the binding of HGF to immobilized shark skin CS/DS chains, suggesting that the pleiotrophin- and HGF-binding domains at least partially overlap in the CS/DS chains involved in the neuritogenic activity. Computational chemistry using molecular modeling and calculations of the electrostatic potential of the hexasaccharide and two pleiotrophin-binding octasaccharides previously isolated from CS/DS hybrid chains of embryonic pig brain identified an electronegative zone potentially involved in the molecular recognition of the oligosaccharides by pleiotrophin. Homology modeling of pleiotrophin based on a related midkine protein structure predicted the binding pocket of pleiotrophin for the oligosaccharides and provided new insights into the molecular mechanism of the interactions between the oligosaccharides and pleiotrophin.

Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid.

The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane alpha-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.

Two related but distinct chondroitin sulfate mimetope octasaccharide sequences recognized by monoclonal antibody WF6.

Chondroitin sulfate (CS) proteoglycans are major components of cartilage and other connective tissues. The monoclonal antibody WF6, developed against embryonic shark cartilage CS, recognizes an epitope in CS chains, which is expressed in ovarian cancer and variably in joint diseases. To elucidate the structure of the epitope, we isolated oligosaccharide fractions from a partial chondroitinase ABC digest of shark cartilage CS-C and established their chain length, disaccharide composition, sulfate content, and sulfation pattern. These structurally defined oligosaccharide fractions were characterized for binding to WF6 by enzyme-linked immunosorbent assay using an oligosaccharide microarray prepared with CS oligosaccharides derivatized with a fluorescent aminolipid. The lowest molecular weight fraction recognized by WF6 contained octasaccharides, which were split into five subfractions. The most reactive subfraction contained several distinct octasaccharide sequences. Two octasaccharides, DeltaD-C-C-C and DeltaC-C-A-D (where A represents GlcUAbeta1-3GalNAc(4-O-sulfate), C is GlcUAbeta1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate), DeltaCis Delta(4,5)HexUAalpha1-3GalNAc(6-O-sulfate), and DeltaDis Delta(4,5)HexUA(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate)), were recognized by WF6, but other related octasaccharides, DeltaC-A-D-C and DeltaC-C-C-C, were not. The structure and sequences of both the binding and nonbinding octasaccharides were compared by computer modeling, which revealed a remarkable similarity between the shape and distribution of the electrostatic potential in the two different octasaccharide sequences that bound to WF6 and that differed from the nonbinding octasaccharides. The strong similarity in structure predicted for the two binding CS octasaccharides (DeltaD-C-C-C and DeltaC-C-A-D) provided a possible explanation for their similar affinity for WF6, although they differed in sequence and thus form two specific mimetopes for the antibody.

Suppression of IL-6 production and proliferation by blocking STAT3 activation in malignant soft tissue tumor cells.

There is no established optimum treatment for malignant fibrous histiocytoma (MFH) at present, and few MFH cell lines are established. In the present study, we established new MFH cell lines, KHZ-MFH and SFT85-03, and investigated the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway. We found that MFH cells secreted high levels of IL-6 and that STAT3 was constitutively activated in these cells. The JAK2 kinase inhibitor, tyrphostin AG490, suppressed the growth of MFH cells and inhibited the secretion of IL-6. Furthermore, blockade of activated STAT3 by forced expression of a cytokine signaling repressor, SOCS3 gene as well as a dominant-negative STAT3 in these cells significantly suppressed their growth. These results indicated that an autocrine mechanism of the JAK/STAT3 signaling pathway could promote the growth of MFH cells and that this pathway could be a therapeutic target of MFH.

Ghrelin directly regulates bone formation.

UNLABELLED: To clarify the role of ghrelin in bone metabolism, we examined the effect of ghrelin in vitro and in vivo. Ghrelin and its receptor, GHS-R1a, were identified in osteoblasts, and ghrelin promoted both proliferation and differentiation. Furthermore, ghrelin increased BMD in rats. Our results show that ghrelin directly affects bone formation. INTRODUCTION: Ghrelin is a gut peptide involved in growth hormone (GH) secretion and energy homeostasis. Recently, it has been reported that the adipocyte-derived hormone leptin, which also regulates energy homeostasis and opposes ghrelin's actions in energy homeostasis, plays a significant role in bone metabolism. This evidence implies that ghrelin may modulate bone metabolism; however, it has not been clarified. To study the role of ghrelin in skeletal integrity, we examined its effects on bone metabolism both in vitro and in vivo. MATERIALS AND METHODS: We measured the expression of ghrelin and growth hormone secretagogue receptor 1a (GHS-R1a) in rat osteoblasts using RT-PCR and immunohistochemistry (IHC). The effect of ghrelin on primary osteoblast-like cell proliferation was examined by recording changes in cell number and the level of DNA synthesis. Osteoblast differentiation markers (Runx2, collagen alpha1 type I [COLI], alkaline phosphatase [ALP], osteocalcin [OCN]) were analyzed using quantitative RT-PCR. We also examined calcium accumulation and ALP activity in osteoblast-like cells induced by ghrelin. Finally, to address the in vivo effects of ghrelin on bone metabolism, we examined the BMD of Sprague-Dawley (SD) rats and genetically GH-deficient, spontaneous dwarf rats (SDR). RESULTS: Ghrelin and GHS-R1a were identified in osteoblast-like cells. Ghrelin significantly increased osteoblast-like cell numbers and DNA synthesis in a dose-dependent manner. The proliferative effects of ghrelin were suppressed by [D-Lys(3)]-GHRP-6, an antagonist of GHS-R1a, in a dose-dependent manner. Furthermore, ghrelin increased the expression of osteoblast differentiation markers, ALP activity, and calcium accumulation in the matrix. Finally, ghrelin definitely increased BMD of both SD rats and SDRs. CONCLUSIONS: These observations show that ghrelin directly stimulates bone formation.

Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway.

Neuromedin U (NMU) is a hypothalamic neuropeptide that regulates body weight and composition. Here we show that mice lacking the gene encoding NMU (Nmu(-/-) mice) develop obesity. Nmu(-/-) mice showed increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese Nmu(-/-) mice developed hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia and hyperlipidemia. Notably, however, treatment with exogenous leptin was effective in reducing body weight in obese Nmu(-/-) mice. In addition, central leptin administration did not affect NMU gene expression in the hypothalamus of rats. These results indicate that NMU plays an important role in the regulation of feeding behavior and energy metabolism independent of the leptin signaling pathway. These characteristic functions of NMU may provide new insight for understanding the pathophysiological basis of obesity.

The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system.

Immunohistochemical analysis revealed the presence of a gut-brain peptide, neuromedin U (NMU), in the suprachiasmatic nucleus (SCN), which is the site of the master circadian oscillator. The expression of NMU mRNA exhibited a circadian rhythm, with the peak expression in the SCN occurring at CT4-8h. The two NMU-binding receptors (NMU-R1 and NMU-R2) were also expressed in the SCN, but their phase angles were different. Intracerebroventricular injection (ICV) of NMU induced the expression of Fos protein in the SCN cells and caused a phase-dependent phase shift of the circadian locomotor activity rhythm. The magnitude of the phase shift was dose dependent. This NMU-induced phase shift was of the nonphotic type. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed increases in the expression in the SCN of immediate early genes, such as c-fos, NGFI-A, NGFI-B, and JunB. Furthermore, ICV injection of NMU increased the expression of Per1, but not Per2, in the SCN. These results indicate that NMU may play some important role in the circadian oscillator by exerting an autocrine or paracrine action in the SCN.