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  • INFLUENCE OF GLUCOSAMINE ON GLOMERULAR MESEANGIAL CELL TURNOVER: IMPLICATIONS FOR HYPERGLYCEMIA AND HEXOSAMINE PATHWAY FLUX. 19903862

    Cells exposed to high glucose may undergo hypertrophy, proliferation, and apoptosis, but the role of hexosamine flux in mediating these effects has not been fully elucidated. Accordingly, we studied the effects of glucose and glucosamine on rat glomerular mesangial cells (MC) turnover. In comparison with physiologic glucose (5.6mM), treatment with high glucose (25mM) for 24 hours stimulated MC proliferation, an effect that was mimicked by exposure to low concentrations of glucosamine (0.05mM). The percentage of cells in G(0)/G(1) phase of the cell cycle was reduced with a concomitant increase of the number of cells in G2/M phase. Proliferating cell nuclear antigen (PCNA), phosphorylated mammalian target of rapamycin (phospho-mTOR [ser2448]) and total regulatory associated protein of mTOR (Raptor) were increased by high glucose and glucosamine treatment. Inhibition of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for hexosamine flux, with 6-diazo-5-oxonorleucine (DON, 10microM) and of mTOR with rapamycin both attenuated glucose-mediated MC proliferation. Higher glucosamine concentrations (0.25mM to 10mM) caused MC apoptosis after 48 hours and, in addition, GFAT overexpression also increased MC apoptosis (TUNEL-positive cells: 3.8+/-0.3 % versus 1.1+/-0.2% for empty vector; p<0.001). Hence, hexosamine flux is an important determinant of MC proliferation and apoptosis. The proliferative response to high glucose and hexosamine flux is rapamycin-sensitive, suggesting that this effect is associated with signaling through rapamycin-sensitive mTOR complex 1 (mTORC1).
    Document Type:
    Reference
    Product Catalog Number:
    17-500
    Product Catalog Name:
    Catch and Release® v2.0 Reversible Immunoprecipitation System

  • Inhibitory effects of glucosamine on endotoxin-induced uveitis in Lewis rats. 18719082

    Glucosamine sulfate (GS) is a naturally occurring sugar that exerts immunosuppressive effects in vitro and in vivo. The authors investigated whether GS modulates the inflammatory reaction in endotoxin-induced uveitis (EIU) of rats and the mechanisms by which it exerts its effects.Two-hundred micrograms of lipopolysaccharide (LPS) was injected subcutaneously into Lewis rats to induce EIU. Doses of GS (10, 100, or 1000 mg/kg) were divided into three aliquots and administered intraperitoneally 30 minutes before LPS injection, concurrently with LPS injection, and 30 minutes after LPS injection. Twenty-four hours after LPS injection, aqueous humor was collected for cell counting and measurement of protein concentration. Immunohistochemical staining of the iris-ciliary body was performed to evaluate the effects of GS on intercellular adhesion molecule (ICAM)-1 and nuclear factor (NF)-kappaB activation and to demonstrate macrophage infiltration. The effects of various doses of GS pretreatment were also examined using a mouse macrophage cell line (RAW264.7 cells) and LPS stimulation. Levels of prostaglandin (PG)-E2 and nitric oxide (NO) were determined. Expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were measured using Western blot analysis. The effect of GS on LPS-induced NF-kappaB activation in RAW cells was also examined.Cell counting and analysis of protein concentration in aqueous humor revealed that GS suppressed EIU in rats treated with a high dose of GS (1000 mg/kg). Immunohistochemistry showed that treatment with GS reduced ICAM-1 expression and suppressed activation of NF-kappaB in the iris-ciliary body. The main inflammatory cells in the iris-ciliary body during EIU were macrophages. In LPS-stimulated macrophage RAW cell culture, GS inhibited the production of NO and PG-E2, the expression of iNOS and COX-2, and the activation of NF-kappaB.GS suppresses EIU in rats by blockading the NF-kappaB-dependent signaling pathway and the subsequent production of ICAM-1 and proinflammatory mediators. This study has extended the authors' previous observation that GS is a potentially important compound for reducing ICAM-1-mediated inflammatory effects in the eye.
    Document Type:
    Reference
    Product Catalog Number:
    06-573
    Product Catalog Name:
    Anti-iNOS/NOS II Antibody, NT

  • Anti-cancer activity of glucosamine through inhibition of N-linked glycosylation. 24932134

    We have reported that the glucosamine suppressed the proliferation of the human prostate carcinoma cell line DU145 through inhibition of STAT3 signaling. DU145 cells autonomously express IL-6 and the IL-6/STAT3 signaling is activated. IL-6 receptor subunits are subject to N-glycosylation, a posttranslational modification which is important for protein stability and function. We speculated that the inhibition of STAT3 phosphorylation by glucosamine might be a functional consequence of the reduced N-glycosylation of gp130.The human prostate cancer cell lines DU145 and PC-3 and human melanoma cell line A2058 were used in this study. Glucosamine effects on N-glycosylation of glycoproteins were determined by Western blot analysis. IL-6 binding to DU145 cells was analyzed by flow cytometry. The cell proliferation suppression was investigated by colorimetric Janus green staining method.In DU145 cells glucosamine reduced the N-glycosylation of gp130, decreased IL-6 binding to cells and impaired the phosphorylation of JAK2, SHP2 and STAT3. Glucosamine acts in a very similar manner to tunicamycin, an inhibitor of protein N-glycosylation. Glucosamine-mediated inhibition of N-glycosylation was neither protein- nor cell-specific. Sensitivity of DU145, A2058 and PC-3 cells to glucosamine-induced inhibition of N-glycosylation were well correlated to glucosamine cytotoxicity in these cells.Our results suggested that the glucosamine-induced global inhibition of protein N-glycosylation might be the basic mechanism underlying its multiple biochemical and cellular effects.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The inhibitory mechanism of a novel cationic glucosamine derivative against MMP-2 and MMP-9 expressions. 19375915

    A number of recent researches have demonstrated the therapeutic effects of glucosamine (Glc) in a range of human diseases including arthritis. For the first time, we identified that a novel Glc derivative having quaternized amino functionality (QAGlc) suppresses MMP-9 and MMP-2, gelatinases in HT1080, human fibrosarcoma cells at 40microg/ml, following stimulation with PMA. Reporter gene assay results revealed that, the mechanism of suppression involves decreased transcriptional activation of MMP-9 and MMP-2 via transcription factors NF-kappaB and AP-1. However based on western blot results, QAGlc did not attenuate the nuclear translocation of both NF-kappaB and AP-1. Apparently, phorbol myristate acetate (PMA) stimulated expressions of ERK, JNK and p38 were altered in the presence of potent tumour inducer, phorbol myristate acetate QAGlc, suggesting their suppression also contributes to QAGlc-mediated inhibition of MMP-9 and MMP-2. Moreover, the ability of QAGlc to inhibit gelatinases was confirmed by its ability to act against invasiveness of HT1080 cells through extracellular matrix components.
    Document Type:
    Reference
    Product Catalog Number:
    ECM550
    Product Catalog Name:
    QCM ECMatrix Cell Invasion Assay, 24-well (8 µm), colorimetric

  • The heparin/heparan sulfate sequence that interacts with cyclophilin B contains a 3-O-sulfated N-unsubstituted glucosamine residue. 17588944

    Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH2), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH2-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.
    Document Type:
    Reference
    Product Catalog Number:
    MAB2040
    Product Catalog Name:
    Anti-Heparin/Heparan Sulfate Antibody, clone T320.11

  • A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin. 3874872

    A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1685

  • Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis. 19855436

    Heparan sulfate (HS) proteoglycans (HSPGs) bind to multiple growth factors/morphogens and regulate their signaling. 6-O-sulfation (6S) of glucosamine within HS chains is critical for many of these ligand interactions. Sulf-1 and Sulf-2, which are extracellular neutral-pH sulfatases, provide a novel post-synthetic mechanism for regulation of HSPG function by removing 6S from intact HS chains. The Sulfs can thereby modulate several signaling pathways, including the promotion of Wnt signaling. We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small-cell lung carcinomas (NSCLCs). We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas. We studied five Sulf-2(+) NSCLC cell lines, including two, which were derived by cigarette-smoke transformation of bronchial epithelial cells. shRNA-mediated Sulf-2 knockdown in these lines caused an increase in 6S on their cell surface and in parallel reversed their transformed phenotype in vitro, eliminated autocrine Wnt signaling and strongly blunted xenograft tumor formation in nude mice. Conversely, forced Sulf-2 expression in non-malignant bronchial epithelial cells produced a partially transformed phenotype. Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer.
    Document Type:
    Reference
    Product Catalog Number:
    MABC584
    Product Catalog Name:
    Anti-Sulf-2 Antibody, clone 2B4

  • Lacrimal gland development and Fgf10-Fgfr2b signaling are controlled by 2-O- and 6-O-sulfated heparan sulfate. 21357686

    Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.
    Document Type:
    Reference
    Product Catalog Number:
    06-570
    Product Catalog Name:
    Anti-phospho-Histone H3 (Ser10) Antibody, Mitosis Marker

  • Glucose represses connexin36 in insulin-secreting cells. 16263767

    The gap-junction protein connexin36 (Cx36) contributes to control the functions of insulin-producing cells. In this study, we investigated whether the expression of Cx36 is regulated by glucose in insulin-producing cells. Glucose caused a significant reduction of Cx36 in insulin-secreting cell lines and freshly isolated pancreatic rat islets. This decrease appeared at the mRNA and the protein levels in a dose- and time-dependent manner. 2-Deoxyglucose partially reproduced the effect of glucose, whereas glucosamine, 3-O-methyl-D-glucose and leucine were ineffective. Moreover, KCl-induced depolarization of beta-cells had no effect on Cx36 expression, indicating that glucose metabolism and ATP production are not mandatory for glucose-induced Cx36 downregulation. Forskolin mimicked the repression of Cx36 by glucose. Glucose or forskolin effects on Cx36 expression were not suppressed by the L-type Ca(2+)-channel blocker nifedipine but were fully blunted by the cAMP-dependent protein kinase (PKA) inhibitor H89. A 4 kb fragment of the human Cx36 promoter was identified and sequenced. Reporter-gene activity driven by various Cx36 promoter fragments indicated that Cx36 repression requires the presence of a highly conserved cAMP responsive element (CRE). Electrophoretic-mobility-shift assays revealed that, in the presence of a high glucose concentration, the binding activity of the repressor CRE-modulator 1 (CREM-1) is enhanced. Taken together, these data provide evidence that glucose represses the expression of Cx36 through the cAMP-PKA pathway, which activates a member of the CRE binding protein family.
    Document Type:
    Reference
    Product Catalog Number:
    06-519
    Product Catalog Name:
    Anti-phospho-CREB (Ser133) Antibody