Serum and urinary concentrations of heparan sulfate in patients with diabetic nephropathy.
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BACKGROUND: Heparan sulfate (HS) contributes to the negative charge in the glomerular basement membrane (GBM), which may maintain the GBM charge barrier. Changes in sulfation and/or the concentration of HS may be associated with the development of diabetic nephropathy. METHODS: Using two different antibodies specific for HS chains, one that reacts with the N-sulfated sequences in HS chains (10E4) and the other that reacts with neo-epitope of HS, which occurs after heparitinase digestion of HS chains (3G10), we examined the serum and urinary concentrations of HS by enzyme-linked immunosorbent assay and performed immunohistochemical staining of glomeruli in diabetic patients with and without nephropathy. RESULTS: The level of urinary excretion of 10E4 binding HS/creatinine clearance was significantly reduced in diabetic patients when compared with that in nondiabetic subjects (P < 0.0001), and the level was more decreased in patients with overt nephropathy than in patients without overt nephropathy. No differences or only small differences were found between these groups in serum and urinary 3G10-binding HS and in serum 10E4-binding HS. Immunohistochemical staining with these antibodies was consistent with the findings in the urine. CONCLUSIONS: The results suggest that a decreased HS N-sulfation exists in the urine, which may reflect a structural change or an altered processing of HS within the GBM. Because N-sulfation plays a key role in determining the extent of sulfation within the HS chains, the decreased urinary 10E4-binding HS may have potential implications with regard to the development of diabetic nephropathy. (+info)
Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein.
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Applications of gene transfer in acute myeloid leukemia (AML) blast cells have still not been developed, mostly due to the lack of an efficient vector. Adenoviruses have many advantages as vectors, but remain poorly efficient in cells lacking fiber receptors. A promising strategy is the retargeting of adenoviruses to other cellular receptors. We report the dramatic enhancement of gene transfer efficiency in AML blasts using AdZ.F(pK7), a modified adenovirus containing a heparin/heparan sulfate binding domain incorporated into the fiber protein of the adenovirus. We transduced 25 AML blast samples with efficiency reaching 100% of the cells in most samples. Optimal results were obtained at 8400 physical particles per cell, corresponding to a multiplicity of infection of 100 plaque forming units per cell. Control AdZ.F adenovirus efficiently transduced leukemic cell lines but gave poor results in AML samples. Both addition of soluble heparin and cell treatment with heparinase inhibited AdZ.F(pK7) gene transfer, showing that heparan sulfates are the major receptors mediating AdZ.F(pK7) transduction of AML blasts. Although adenoviruses can infect nondividing cells, we observed that a combination of growth factors (GM-CSF, IL-3, stem cell factor) was required for efficient transduction in order to maintain AML blast cell viability. This study demonstrates that retargeting the adenovirus fiber protein to heparan sulfates can overcome the low efficiency of adenovirus in AML blast cells and may provide a useful tool for gene therapy approaches in AML. (+info)
Mapping of a new SGBS locus to chromosome Xp22 in a family with a severe form of Simpson-Golabi-Behmel syndrome.
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Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth syndrome with associated visceral and skeletal abnormalities. Alterations in the glypican-3 gene (GPC3), which is located on Xq26, have been implicated in the etiology of relatively milder cases of this disorder. Not all individuals with SGBS have demonstrated disruptions of the GPC3 locus, which raises the possibility that other loci on the X chromosome could be responsible for some cases of this syndrome. We have previously described a large family with a severe form of SGBS that is characterized by multiple anomalies, hydrops fetalis, and death within the first 8 wk of life. Using 25 simple tandem-repeat polymorphism markers spanning the X chromosome, we have localized the gene for this disorder to an approximately 6-Mb region of Xp22, with a maximum LOD score of 3.31 and with LOD scores <-2.0 for all of Xq. These results demonstrate that neither the GPC3 gene nor other genes on Xq26 are responsible for all cases of SGBS and that a second SGBS locus resides on Xp22. (+info)
Chromatin clearance in C57Bl/10 mice: interaction with heparan sulphate proteoglycans and receptors on Kupffer cells.
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Chromatin is an important autoantigen in the pathogenesis of systemic lupus erythematosus (SLE) as an immunogen and as a part of nephritogenic immune complexes. Earlier studies focused on clearance of DNA. However, DNA released into the circulation from dying cells is found associated with histones in nucleosomes. The liver is the major organ involved in clearance of chromatin from the circulation of mice. Heparan sulphate proteoglycans (HSPG) have been implicated in the clearance of various charged molecules. Receptor-mediated clearance of ssDNA by the liver has also been reported. Because chromatin contains positively charged histones in addition to DNA, we wished to determine if HSPG and/or DNA receptors are involved in chromatin clearance. The rate of clearance of H1-stripped chromatin from the bloodstream of C57Bl/10 mice was markedly decreased by prior treatment of mice with Heparinase I. Clearance was also inhibited by heparin, heparan sulphate, and DNA, but not by colominic acid. DNA was the most effective inhibitor of clearance and released chromatin from sites of clearance. Depletion of Kupffer cells and splenic macrophages using liposome-encapsulated Clodronate (dichloromethylene bisphosphonate) markedly inhibited chromatin clearance. These data suggest that chromatin clearance is mediated by charge interactions with cell surface HSPG and by DNA receptors. Clearance and degradation of chromatin require functional macrophages in the liver and spleen. (+info)
Stromal cell-derived factor-1alpha associates with heparan sulfates through the first beta-strand of the chemokine.
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Biological properties of chemokines are believed to be influenced by their association with glycosaminoglycans. Surface plasmon resonance kinetic analysis shows that the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha), which binds the CXCR4 receptor, associates with heparin with an affinity constant of 38.4 nM (k(on) = 2.16 x 10(6) M(-1) s(-1) and k(off) = 0.083 x s(-1)). A modified SDF-1alpha (SDF-1 3/6) was generated by combined substitution of the basic cluster of residues Lys(24), His(25), and Lys(27) by Ser. SDF-1 3/6 conserves the global native structure and functional properties of SDF-1alpha, but it is unable to interact with sensor chip-immobilized heparin. The biological relevance of these in vitro findings was investigated. SDF-1alpha was unable to bind in a CXCR4-independent manner on epithelial cells that were treated with heparan sulfate (HS)-degrading enzymes or constitutively lack HS expression. The inability of SDF-1 3/6 to bind to cells underlines the importance of the identified basic cluster for the physiological interactions of SDF-1alpha with HS. Importantly, the amino-terminal domain of SDF-1alpha which is required for binding to, and activation of, CXCR4 remains exposed after binding to HS and is recognized by a neutralizing monoclonal antibody directed against the first residues of the chemokine. Overall, these findings indicate that the Lys(24), His(25), and Lys(27) cluster of residues forms, or is an essential part of, the HS-binding site which is distinct from that required for binding to, and signaling through, CXCR4. (+info)
The role of syndecan cytoplasmic domain in basic fibroblast growth factor-dependent signal transduction.
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To determine the role played by syndecan-4 cytoplasmic domain in the mediation of basic fibroblast growth factor (bFGF) signaling, immortalized human cells (ECV) were used to generate cell lines expressing constructs encoding full-length sequences for syndecan-4 (S4), syndecan-1 (S1), glypican-1 (G1), or chimeric proteins consisting of the ectoplasmic domain of glypican-1 linked to the transmembrane/cytoplasmic domain of syndecan-4 (G1-S4c) and the ectoplasmic domain of syndecan-4 linked to the glypican-1 glycosylphosphatidylinositol (GPI) anchor sequence (S4-GPI). Vector-transduced cells (VC) were used as controls. Expression of all these proteoglycans (except for the vector control) significantly increased cell-associated heparan sulfate mass and the number of low affinity bFGF-binding sites. However, in low serum medium, the addition of bFGF stimulated growth and migration of cells expressing S4 and G1-S4c constructs but not G1, S1, S4-GPI, or VC cells. Similar results were obtained using Matrigel growth assays. Mutations of heparan sulfate attachment sites on S4 construct abolished syndecan-4-dependent augmentation of bFGF responses. We conclude that cytoplasmic tail of syndecan-4 plays an important role in bFGF-mediated signal transduction. (+info)
Reduction in glomerular heparan sulfate correlates with complement deposition and albuminuria in active Heymann nephritis.
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In a time-study of active Heymann nephritis, the expression of agrin, the main heparan sulfate proteoglycan in the glomerular basement membrane, was analyzed in relation to deposition of IgG and complement in the glomerular capillary wall and the development of albuminuria. Binding of IgG autoantibodies to the glomerular capillary wall could be detected from 2 wk onward, followed by activation of complement after 6 wk. Progressive albuminuria developed from 6 wk onward to a level of 274+/-68 mg/18 h at week 12. The staining intensity for the agrin core protein decreased slightly, and the staining intensity for the heparan sulfate stubs that were still attached to the core protein after heparitinase digestion remained normal. From week 6 onward, however, a progressive decrease was seen in the staining of two monoclonal antibodies (mAb) directed against different epitopes on the heparan sulfate polysaccharide side chain of agrin (to 35 and 30% of the control level, respectively, at week 12, both mAb P = 0.016). Moreover, albuminuria was inversely correlated with heparan sulfate staining as revealed by these antibodies (r(s) = -0.82 and r(s) = -0.75, respectively, both mAb P < 0.0001). This decrease in heparan sulfate staining was due to a progressive reduction of glomerular heparan sulfate content to 46 and 32% of control level at week 10 and week 12 of the disease, respectively, as measured biochemically. It is speculated that the observed decrease in glomerular heparan sulfate in active Heymann nephritis is due to complement-mediated cleavage of heparan sulfate, resulting in an increased permeability of the glomerular basement membrane to macromolecules. (+info)
Sulfated polysaccharide-directed recruitment of mammalian host proteins: a novel strategy in microbial pathogenesis.
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Fundamental to the virulence of microbial pathogens is their capacity for adaptation and survival within variable, and often hostile, environments encountered in the host. We describe a novel, extragenomic mechanism of surface modulation which may amplify the adaptive and pathogenic potential of numerous bacterial species, including Staphylococcus, Yersinia, and pathogenic Neisseria species, as well as Helicobacter pylori and Streptococcus pyogenes. The mechanism involves specific bacterial recruitment of heparin, glycosaminoglycans, or related sulfated polysaccharides, which in turn serve as universal binding sites for a diverse array of mammalian heparin binding proteins, including adhesive glycoproteins (vitronectin and fibronectin), inflammatory (MCP-3, PF-4, and MIP-1alpha) and immunomodulatory (gamma interferon) intermediates, and fibroblast growth factor. This strategy impacts key aspects of microbial pathogenicity as exemplified by increased bacterial invasion of epithelial cells and inhibition of chemokine-induced chemotaxis. Our findings illustrate a previously unrecognized form of parasitism that complements classical virulence strategies encoded within the microbial genome. (+info)