A novel oligosaccharide, GlcA beta 1-4Xyl beta 1-(4-methylumbelliferone), synthesized by human cultured skin fibroblasts.
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ABSTRACT:
SUBMITTER: Nakamura T
PROVIDER: S-EPMC1137395 | biostudies-other | 1994 Dec
REPOSITORIES: biostudies-other
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Project description:Cultured skin fibroblasts established from goats affected with beta-mannosidosis, an inherited neurovisceral storage disorder, showed an absence of lysosomal beta-mannosidase activity and the corresponding accumulation of a trisaccharide (TS) with the structure Man beta (1----4)GlcNAc beta (1----4)GlcNAc (0.4 mumol/g) and lesser amounts (0.15 mumol/g) of a Man beta (1----4)GlcNAc disaccharide (DS). By using purified storage TS isolated from fibroblasts metabolically labelled with [3H]GlcN, no conversion of TS into DS could be demonstrated in homogenates of affected cells at either lysosomal pH (4.4) or cytosolic pH (6.1), or in the culture medium (pH 7.0) of affected cells. Both TS and DS were secreted into the culture medium by affected fibroblasts. When affected fibroblasts were treated with tunicamycin before labelling with [3H]GlcN, the accumulation of both labelled TS and DS was completely inhibited. Treatment of both affected and normal goat fibroblasts with swainsonine resulted in the inhibition of lysosomal alpha-mannosidase activity and in the accumulation of the same labelled oligosaccharides in both. The major storage pentasaccharide from both normal and affected swainsonine-treated fibroblasts was sensitive to digestion with alpha-mannosidase and endo-beta-N-acetylhexosaminidase D, suggesting a branched mannose structure and a chitobiose core. In the absence of evidence for the existence of unusual N-linked glycoprotein-associated chitotriose oligosaccharide structures in affected goat fibroblasts, it must be concluded that degradative pathways for N-linked oligosaccharides are similar in both normal and affected goat fibroblasts, and that these pathways differ from catabolic pathways in human fibroblasts.
| S-EPMC1146542 | biostudies-other
Project description:A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.
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Project description:Insulin-like growth factor 1 (IGF-1) stimulates cell proliferation and is crucial for maintenance of somatic tissues. However, this effect is associated with the inhibition of FOXO transcription factors and downregulation of antioxidative enzymes. In this study, we compared the responses of primary dermal fibroblasts and human umbilical vein endothelial cells with IGF-1 treatment. We found that IGF-1 primarily downregulated enzymatic antioxidants in skin fibroblasts. However, human umbilical vein endothelial cells were protected from an IGF-1-mediated decrease in antioxidative capacity. Moreover, IGF-1 also activated endothelial nitric oxide synthase in human umbilical vein endothelial cells. These observations suggest a dichotomous role for IGF-1, which provides for growth and repair needs of the soma, while attenuating the effect of oxidative stress on the vasculature by activating endothelial nitric oxide synthase. This increases the production of nitric oxide, an antiproliferative and, under certain circumstances, an antioxidant agent. Findings could help clarify the role of IGF-1 in aging and longevity of lower organisms, short-lived mammals, and humans.
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Project description:Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms.We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR.Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 x 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status). An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater.
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Project description:1. Human skin fibroblasts internalize homologous sulphated proteoglycans by adsorptive endocytosis. Endocytosis rate is half maximal when the concentration of the proteoglycans is 0.1 nM. At saturation, a single fibroblast may endocytose up to 8 X 10(6) proteoglycan molecules/h. 2. The kinetics of prote;glycan binding to the cell surface suggest the presence of 6 X 10(5) high-affinity binding sites per cell. The bulk of sulphated proteoglycans associates to low-affinity binding sites on the cell surface. 3. Glycosaminoglycans and other anionic macromolecules inhibit endocytosis of sulphated proteoglycans non-competitively. The lack of interaction of glycosaminoglycans with the cell-surface receptors for sulphated proteoglycans suggests that the protein core of proteoglycans is essential for binding to the cell surface. 4. The effects of trypsin, cell density, serum concentration and medium pH on endocytosis and degradation of endocytosed sulphated proteoglycans is described. 5. A comparison of the number of the high-affinity binding sites and the number of molecules endocytosed with respect to time suggests a recycling of the proteoglycan receptors between the cell surface and the endocytotic vesicles and/or the lysosomes.
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Project description:The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase.
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Project description:Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.
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Project description:Skin responds to environmental stressors via coordinated actions of the local neuroimmunoendocrine system. Although some of these responses involve opioid receptors, little is known about cutaneous proenkephalin expression, its environmental regulation, and alterations in pathology. The objective of this study was to assess regulated expression of proenkephalin in normal and pathological skin and in isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells. The proenkephalin gene and protein were expressed in skin and cultured cells, with significant expression in fibroblasts and keratinocytes. Mass spectroscopy confirmed Leu- and Met-enkephalin in skin. UVR, Toll-like receptor (TLR)4, and TLR2 agonists stimulated proenkephalin gene expression in melanocytes and keratinocytes in a time- and dose-dependent manner. In situ Met/Leu-enkephalin peptides were expressed in differentiating keratinocytes of the epidermis in the outer root sheath of the hair follicle, in myoepithelial cells of the eccrine gland, and in the basement membrane/basal lamina separating epithelial and mesenchymal components. Met/Leu-enkephalin expression was altered in pathological skin, increasing in psoriasis and decreasing in melanocytic tumors. Not only does human skin express proenkephalin, but this expression is upregulated by stressful stimuli and can be altered by pathological conditions.
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