| 1. |
- Jönsson, Mats, et al.
(author)
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Initiation of the decorin glycosaminoglycan chain in the endoplasmic reticulum-Golgi intermediate compartment
- 2003
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In: Journal of Biological Chemistry. - 1083-351X. ; 278:24, s. 21415-21420
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Journal article (peer-reviewed)abstract
- We have transiently expressed decorin with a C- terminal KDEL endoplasmic reticulum retention signal peptide in COS- 7 cells to study initiation of galactosaminoglycan synthesis in the endoplasmic reticulum- Golgi intermediate compartment. All decorin- KDEL molecules were substituted with N- linked oligosaccharides sensitive to endoglycosidase H, indicating that the core protein was located proximal to the medial- Golgi. O-Linked glycosylation was only initiated in a minor fraction of the molecules. The O- linked saccharides were characterized by gel filtration after stepwise degradations using chondroitin ABC/ AC-I lyases, beta1 - 3- glycuronidase, beta-galactosidase, and alkaline phosphatase. The major O- linked saccharide was the linkage region pentasaccharide GalNAcbeta1-4GlcUAbeta1-3Galbeta1-3Galbeta1-4-Xyl- 2- phosphate, demonstrating initiation of chondroitin synthesis in the endoplasmic reticulum- Golgi intermediate compartment. In the presence of brefeldin A, partial elongation of a chondroitin chain took place, indicating retrieval of polymerases but not of sulfotransferases.
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| 2. |
- Belting, Mattias, et al.
(author)
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Glypican-1 is a vehicle for polyamine uptake in mammalian cells. A pivotal role for nitrosothiol-derived nitric oxide.
- 2003
-
In: Journal of Biological Chemistry. - 1083-351X. ; 278:47, s. 47181-47189
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Journal article (peer-reviewed)abstract
- Polyamines (putrescine, spermidine, and spermine) are essential for growth and survival of all cells. When polyamine biosynthesis is inhibited, there is up-regulation of import. The mammalian polyamine transport system is unknown. We have previously shown that the heparan sulfate (HS) side chains of recycling glypican-1 (Gpc-1) can sequester spermine, that intracellular polyamine depletion increases the number of NO-sensitive N-unsubstituted glucosamines in HS, and that NO-dependent cleavage of HS at these sites is required for spermine uptake. The NO is derived from S-nitroso groups in the Gpc-1 protein. Using RNA interference technology as well as biochemical and microscopic techniques applied to both normal and uptake-deficient cells, we demonstrate that inhibition of Gpc-1 expression abrogates spermine uptake and intracellular delivery. In unperturbed cells, spermine and recycling Gpc-1 carrying HS chains rich in N-unsubstituted glucosamines were co-localized. By exposing cells to ascorbate, we induced release of NO from the S-nitroso groups, resulting in HS degradation and unloading of the sequestered polyamines as well as nuclear targeting of the deglycanated Gpc-1 protein. Polyamine uptake-deficient cells appear to have a defect in the NO release mechanism. We have managed to restore spermine uptake partially in these cells by providing spermine NONOate and ascorbate. The former bound to the HS chains of recycling Gpc-1 and S-nitrosylated the core protein. Ascorbate released NO, which degraded HS and liberated the bound spermine. Recycling HS proteoglycans of the glypican-type may be plasma membrane carriers for cargo taken up by caveolar endocytosis.
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| 3. |
- Belting, Mattias, et al.
(author)
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Tumor attenuation by combined heparan sulfate and polyamine depletion.
- 2002
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In: Proceedings of the National Academy of Sciences. - 1091-6490. ; 99:1, s. 371-376
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Journal article (peer-reviewed)abstract
- Cells depend on polyamines for growth and their depletion represents a strategy for the treatment of cancer. Polyamines assemble de novo through a pathway sensitive to the inhibitor, alpha-difluoromethylornithine (DFMO). However, the presence of cell-surface heparan sulfate proteoglycans may provide a salvage pathway for uptake of circulating polyamines, thereby sparing cells from the cytostatic effect of DFMO. Here we show that genetic or pharmacologic manipulation of proteoglycan synthesis in the presence of DFMO inhibits cell proliferation in vitro and in vivo. In cell culture, mutant cells lacking heparan sulfate were more sensitive to the growth inhibitory effects of DFMO than wild-type cells or mutant cells transfected with the cDNA for the missing biosynthetic enzyme. Moreover, extracellular polyamines did not restore growth of mutant cells, but completely reversed the inhibitory effect of DFMO in wild-type cells. In a mouse model of experimental metastasis, DFMO provided in the water supply also dramatically diminished seeding and growth of tumor foci in the lungs by heparan sulfate-deficient mutant cells compared with the controls. Wild-type cells also formed tumors less efficiently in mice fed both DFMO and a xylose-based inhibitor of heparan sulfate proteoglycan assembly. The effect seemed to be specific for heparan sulfate, because a different xyloside known to affect only chondroitin sulfate did not inhibit tumor growth. Hence, combined inhibition of heparan sulfate assembly and polyamine synthesis may represent an additional strategy for cancer therapy.
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| 4. |
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| 5. |
- Buga, Sergei G., et al.
(author)
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Dissociation energy of 3D-polymeric C60 : Calorimetric study and structural analysis
- 2001
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In: Electronic Properties of Novel Materials - Science and Technology of Molecular Nanostructures. - Melville, NY : American Institute of Physics. - 0735400334 ; , s. 49-53
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Conference paper (peer-reviewed)abstract
- Annealing of 2D- and 3D-polymeric C[sub60] fullerene obtained under pressures of 9.5 and 12.5 GPa and temperatures of 670 and 770 K has been investigated by DSC in the range 240–640 K. An endothermal heat effect was observed with a peak maximum just below 540 K, a temperature characteristic for breakdown of (2+2) intermolecular links in dimers, 1D and 2D polymers. Exothermal effects, starting from 380 K, were observed for the first time in polymeric fullerenes. These effects are attributed to relaxation processes and to breakdown of other types of intermolecular bonds such as common four-sided rings and (3+3) interlinks.
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| 6. |
- Buga, Sergei G., et al.
(author)
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DSC study of annealing and phase transformations of C60 and C70 polymerized under pressures in the range 9.5 - 13 GPa.
- 2002
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In: Journal of Physics and Chemistry of Solids. - : Elsevier B.V.. - 0022-3697 .- 1879-2553. ; 63:2, s. 331-343
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Journal article (peer-reviewed)abstract
- C60 and C70 fullerenes polymerized under pressures between 9.5 and 13 GPa and temperatures between 670 and 1850 K were investigated by differential scanning calorimetry (DSC) in the range 240–640 K. Endothermal heat effects were observed with a peak maximum just below 540 K, a temperature characteristic for breakdown of (2+2) intermolecular links in dimers, 1D and 2D polymers. Exothermal effects, starting from 380 K, were observed for the first time in polymeric fullerenes. These effects are attributed to relaxation processes and to breakdown of other types of intermolecular bonds such as common four-sided rings and (3+3) interlinks.
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| 7. |
- Buga, Sergei G., et al.
(author)
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Synthesis of superhard 3D-polymeric C60 fullerites from rhombohedral 2D-polymer by high-pressure-high-temperature treatment.
- 2003
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In: High Pressure Research vol. 23, issue 3. - London : Taylor & Francis. ; , s. 259-264
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Conference paper (peer-reviewed)abstract
- Rhombohedral C60 polymer was subjected to high-pressure-high-temperature treatment at P =13 GPa, T =620-1620 K. After quenching, crystalline and disordered structures with densities in the range of 2.1-2.9 g cm-1 were obtained. The structures of the samples have been investigated by powder X-ray diffraction and Raman scattering. DSC analysis showed a transformation of the polymeric structure into monomeric on annealing in the range 400-640 K. The temperature dependence of the electrical resistance of samples with disordered structure was measured in the range 2.5-300K. For different samples, the conductivity was proportional to T1/2, T3/2, T4 and exp(-1/T1/4).
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| 8. |
- Buga, Sergei, et al.
(author)
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Thermal stability of superhard and ultrahard fullerites
- 2000
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In: Proceedings of International Conference on Engineering and Technological Sciences 2000 (ICETS-2000), vol. 1. - Beijng : Chinese Academy of Engineering: New World Press. ; , s. 505-511
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Conference paper (peer-reviewed)abstract
- The thermal stability of superhard and ultrahard carbon phases synthesized from C60 and C70 fullerenes under pressure in the range 9.5-13 GPa and temperature between 670 and 1850 K was investigated by DSC in the range 260-640 K and TGA up to 1200 K. Endothermal heat effects were observed with a peak maximum just below 540 K, a temperature characteristic for breakdown of (2+2) intermolecular links in dimers, 1D and 2D polymers. Exothermal effects, starting from 380 K, were observed for the first time in polymeric fullerenes. These effects are attributed to relaxation processes and to breakdown of other types of intermolecular bonds.
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| 9. |
- Cheng, Fang, et al.
(author)
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Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1 containing endosomes.
- 2002
-
In: Journal of Biological Chemistry. - 1083-351X. ; 277:46, s. 44431-44439
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Journal article (peer-reviewed)abstract
- We have previously demonstrated intracellular degradation of the heparan sulfate side-chains in recycling glypican-1 by heparanase and by deaminative cleavage at N-unsubstituted glucosamine with nitric oxide derived from intrinsic nitrosothiols [see Ding, K., Mani, K., Cheng, F., Belting, M. and Fransson, L.-. (2002) J. Biol. Chem., 277, xxx-xxx; prepub M203383200]. To determine where and in what order events take place, we have visualized, by using confocal laser-scanning immunofluorescence microscopy, glypican-1 variants in unperturbed cells or arrested at various stages of processing. In unperturbed proliferating cells, glypican-1 was partly S-nitrosylated. Intracellular glypican-1 was enriched in endosomes, colocalized significantly with GM-1 ganglioside, caveolin-1 and Rab9-positive endosomes, and carried side-chains rich in N-unsubstituted glucosamine residues. However, such residues were scarce in cell-surface glypican-1. Brefeldin A-arrested glypican-1, which was non-S-nitrosylated and carried side-chains rich in N-unsubstituted glucosamines, colocalized extensively with caveolin-1 but not with Rab9. Suramin, which inhibits heparanase, induced the appearance of S-nitrosylated glypican-1 in caveolin-1-rich compartments. Inhibition of deaminative cleavage did not prevent heparanase from generating heparan sulfate oligosaccharides that colocalized strongly with caveolin-1. Growth-quiescent cells displayed extensive NO-dependent deaminative cleavage of heparan sulfate generating anhydromannose-terminating fragments which were partly associated with acidic vesicles. Proliferating cells generated such fragments during polyamine uptake. We conclude that recycling glypican-1 that is associated with caveolin-1-containing endosomes undergoes sequential N-desulfation/N-deacetylation, heparanase cleavage, S-nitrosylation, NO-release and deaminative cleavage of its side-chains in conjunction with polyamine uptake.
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| 10. |
- Ding, Kan, et al.
(author)
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Copper-dependent autocleavage of glypican-1heparan sulfate by nitric oxide derived fromintrinsic nitrosothiols.
- 2002
-
In: Journal of Biological Chemistry. - 1083-351X. ; 277:36, s. 33353-33360
-
Journal article (peer-reviewed)abstract
- Cell-surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines [ [Belting, M., Borsig, L., Fuster, M.M., Brown, J.R., Persson, L., Fransson,L.-. and Esko, J.D. (2002) Proc. Natl. Acad. Sci. U.S.A., 99, 371-376] ]. Increased polyamine uptake correlates with an increased number of positively charged N-unsubstituted glucosamine units in the otherwise polyanionic heparan sulfate chains of glypican-1. During intracellular recycling of glypican-1 there is an NO-dependent deaminative cleavage of heparan sulfate at these glucosamine units, which would eliminate the positive charges [ [Ding, K., Sandgren, S., Mani, K., Belting, M. and Fransson, L.-. (2001) J. Biol. Chem., 276, 46779-46791] ]. Here, using both biochemical and microscopic techniques, we have identified and isolated S-nitrosylated forms of glypican-1 as well as low-charged glypican-1 glycoforms containing heparan sulfate chains rich in N-unsubstituted glucosamines. The latter were converted to high-charged species upon treatment of cells with 1 mM L-ascorbate, which releases NO from nitrosothiols, resulting in deaminative cleavage of heparan sulfate at the N-unsubstituted glucosamines. S-nitrosylation and subsequent deaminative cleavage were abrogated by inhibition of a Cu 2+ /Cu + -redox cycle. Under cell-free conditions, purified, S-nitrosylated glypican-1 was able to autocleave its heparan sulfate chains when NO-release was triggered by L-ascorbate. The heparan sulfate fragments generated in cells during this auto-catalytic process contained terminal anhydromannose residues. We conclude that the core protein of glypican-1 can slowly accumulate NO as nitrosothiols while Cu 2+ is reduced to Cu +. Subsequent release of NO results in efficient deaminative cleavage of the heparan sulfate chains attached to the same core protein while Cu + is oxidized to Cu 2+.
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