Chondroitin sulfate effects on neural stem cell differentiation.

Science.gov (United States)

Canning, David R; Brelsford, Natalie R; Lovett, Neil W

2016-01-01

We have investigated the role chondroitin sulfate has on cell interactions during neural plate formation in the early chick embryo. Using tissue culture isolates from the prospective neural plate, we have measured neural gene expression profiles associated with neural stem cell differentiation. Removal of chondroitin sulfate from stage 4 neural plate tissue leads to altered associations of N-cadherin-positive neural progenitors and causes changes in the normal sequence of neural marker gene expression. Absence of chondroitin sulfate in the neural plate leads to reduced Sox2 expression and is accompanied by an increase in the expression of anterior markers of neural regionalization. Results obtained in this study suggest that the presence of chondroitin sulfate in the anterior chick embryo is instrumental in maintaining cells in the neural precursor state.

Heparan sulfate-chondroitin sulfate hybrid proteoglycan of the cell surface and basement membrane of mouse mammary epithelial cells

International Nuclear Information System (INIS)

David, G.; Van den Berghe, H.

1985-01-01

Chondroitin sulfate represents approximately 15% of the 35 SO 4 -labeled glycosaminoglycans carried by the proteoglycans of the cell surface and of the basolateral secretions of normal mouse mammary epithelial cells in culture. Evidence is provided that these chondroitin sulfate-carrying proteoglycans are hybrid proteoglycans, carrying both chondroitin sulfate and heparan sulfate chains. Complete N-desulfation but limited O-desulfation, by treatment with dimethyl sulfoxide, of the proteoglycans decreased the anionic charge of the chondroitin sulfate-carrying proteoglycans to a greater extent than it decreased the charge of their constituent chondroitin sulfate chains. Partial depolymerization of the heparan sulfate residues of the proteoglycans with nitrous acid or with heparin lyase also reduced the effective molecular radius of the chondroitin sulfate-carrying proteoglycans. The effect of heparin lyase on the chondroitin sulfate-carrying proteoglycans was prevented by treating the proteoglycan fractions with dimethyl sulfoxide, while the effect of nitrous acid on the dimethyl sulfoxide-treated proteoglycans was prevented by acetylation. This occurrence of heparan sulfate-chondroitin sulfate hybrid proteoglycans suggests that the substitution of core proteins by heparan sulfate or chondroitin sulfate chains may not solely be determined by the specific routing of these proteins through distinct chondroitin sulfate and heparan sulfate synthesizing mechanisms. Moreover, regional and temporal changes in pericellular glycosaminoglycan compositions might be due to variable postsynthetic modification of a single gene product

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

International Nuclear Information System (INIS)

Silbert, C.K.; Humphries, D.E.; Palmer, M.E.; Silbert, J.E.

1991-01-01

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics

The effect of divalent salt in chondroitin sulfate solutions

Energy Technology Data Exchange (ETDEWEB)

Aranghel, D., E-mail: daranghe@nipne.ro [Horia Hulubei National Institute of Physics and Nuclear Engineering, Reactorului 30, RO-077125, POB-MG6, Magurele-Bucharest, Romania, daranghe@nipne.ro (Romania); Extreme Light Intrastructure Nuclear Physics (ELI-NP), Reactorului 30,RO-077125, POB-MG6, Magurele-Bucharest (Romania); Badita, C. R. [Horia Hulubei National Institute of Physics and Nuclear Engineering, Reactorului 30, RO-077125, POB-MG6, Magurele-Bucharest, Romania, daranghe@nipne.ro (Romania); University of Bucharest, Faculty of Physics, Atomiştilor 405, CP MG - 11, RO – 077125, Bucharest-Magurele (Romania); Radulescu, A. [Forschungszentrum Jülich GmbH, Jülich Centre for Neutron Science, 85747 Garching (Germany); Moldovan, L.; Craciunescu, O. [National Institute R& D for Biological Sciences, Splaiul Independenţei 296, sector 6, cod 060031, C.P. 17-16, Bucharest (Romania); Balasoiu, M. [Horia Hulubei National Institute of Physics and Nuclear Engineering, Reactorului 30, RO-077125, POB-MG6, Magurele-Bucharest, Romania, daranghe@nipne.ro (Romania); Joint Institute for Nuclear Research, 141980 Dubna, Moscow region (Russian Federation)

2016-03-25

Chondroitin-4 sulfate (CS4) is the main glycosaminoglycan extracted from bovine trachea. CS4 play an important role in osteoarthritis treatment, anticoagulant activity, reduces the degradation of cartilage matrix components, reduces necrosis and apoptosis of chondrocytes and reduces the activity of collagenase. Chondroitin sulfate is also responsible for proteoglycans degradation. Chondroitin sulfate can bind calcium ions with different affinities, depending on their sulfation position. The purpose of this study was to determine the structural properties and the influence of Ca{sup 2+} cations. We carried out measurements on CS4 solutions and mixtures of liquid CS4 with Ca{sup 2+} by Small-Angle Neutron Scattering (SANS). CS4 have a mass fractal behavior and the addition of a salt (CaCl{sub 2}) in CS4 solutions generates the appearance of a correlation peak due to local ordering between adjacent chains with inter-chain distances between 483 Å and 233 Å for a calcium concentration of 0.01% w/w.

The effect of divalent salt in chondroitin sulfate solutions

Science.gov (United States)

Aranghel, D.; Badita, C. R.; Radulescu, A.; Moldovan, L.; Craciunescu, O.; Balasoiu, M.

2016-03-01

Chondroitin-4 sulfate (CS4) is the main glycosaminoglycan extracted from bovine trachea. CS4 play an important role in osteoarthritis treatment, anticoagulant activity, reduces the degradation of cartilage matrix components, reduces necrosis and apoptosis of chondrocytes and reduces the activity of collagenase. Chondroitin sulfate is also responsible for proteoglycans degradation. Chondroitin sulfate can bind calcium ions with different affinities, depending on their sulfation position. The purpose of this study was to determine the structural properties and the influence of Ca2+ cations. We carried out measurements on CS4 solutions and mixtures of liquid CS4 with Ca2+ by Small-Angle Neutron Scattering (SANS). CS4 have a mass fractal behavior and the addition of a salt (CaCl2) in CS4 solutions generates the appearance of a correlation peak due to local ordering between adjacent chains with inter-chain distances between 483 Å and 233 Å for a calcium concentration of 0.01% w/w.

The effect of divalent salt in chondroitin sulfate solutions

International Nuclear Information System (INIS)

Aranghel, D.; Badita, C. R.; Radulescu, A.; Moldovan, L.; Craciunescu, O.; Balasoiu, M.

2016-01-01

Chondroitin-4 sulfate (CS4) is the main glycosaminoglycan extracted from bovine trachea. CS4 play an important role in osteoarthritis treatment, anticoagulant activity, reduces the degradation of cartilage matrix components, reduces necrosis and apoptosis of chondrocytes and reduces the activity of collagenase. Chondroitin sulfate is also responsible for proteoglycans degradation. Chondroitin sulfate can bind calcium ions with different affinities, depending on their sulfation position. The purpose of this study was to determine the structural properties and the influence of Ca"2"+ cations. We carried out measurements on CS4 solutions and mixtures of liquid CS4 with Ca"2"+ by Small-Angle Neutron Scattering (SANS). CS4 have a mass fractal behavior and the addition of a salt (CaCl_2) in CS4 solutions generates the appearance of a correlation peak due to local ordering between adjacent chains with inter-chain distances between 483 Å and 233 Å for a calcium concentration of 0.01% w/w.

Structural elucidation of fucosylated chondroitin sulfates from sea cucumber using FTICR-MS/MS.

Science.gov (United States)

Agyekum, Isaac; Pepi, Lauren; Yu, Yanlei; Li, Junhui; Yan, Lufeng; Linhardt, Robert J; Chen, Shiguo; Amster, I Jonathan

2018-02-01

Fucosylated chondroitin sulfates are complex polysaccharides extracted from sea cucumber. They have been extensively studied for their anticoagulant properties and have been implicated in other biological activities. While nuclear magnetic resonance spectroscopy has been used to extensively characterize fucosylated chondroitin sulfate oligomers, we herein report the first detailed mass characterization of fucosylated chondroitin sulfate using high-resolution Fourier transform ion cyclotron resonance mass spectrometry. The two species of fucosylated chondroitin sulfates considered for this work include Pearsonothuria graeffei (FCS-Pg) and Isostichopus badionotus (FCS-Ib). Fucosylated chondroitin sulfate oligosaccharides were prepared by N-deacetylation-deaminative cleavage of the two fucosylated chondroitin sulfates and purified by repeated gel filtration. Accurate mass measurements obtained from electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry measurements confirmed the oligomeric nature of these two fucosylated chondroitin sulfate oligosaccharides with each trisaccharide repeating unit averaging four sulfates per trisaccharide. Collision-induced dissociation of efficiently deprotonated molecular ions through Na/H + exchange proved useful in providing structurally relevant glycosidic and cross-ring product ions, capable of assigning the sulfate modifications on the fucosylated chondroitin sulfate oligomers. Careful examination of the tandem mass spectrometry of both species deferring in the positions of sulfate groups on the fucose residue (FCS-Pg-3,4- OS) and (FCS-Ib-2,4- OS) revealed cross-ring products 0,2 A αf and 2,4 X 2αf which were diagnostic for (FCS-Pg-3,4- OS) and 0,2 X 2αf diagnostic for (FCS-Ib-2,4- OS). Mass spectrometry and tandem mass spectrometry data acquired for both species varying in oligomer length (dp3-dp15) are presented.

Development of affinity-based delivery of NGF from a chondroitin sulfate biomaterial.

Science.gov (United States)

Butterfield, Karen Chao; Conovaloff, Aaron W; Panitch, Alyssa

2011-01-01

Chondroitin sulfate is a major component of the extracellular matrix in both the central and peripheral nervous systems. Chondroitin sulfate is upregulated at injury, thus methods to promote neurite extension through chondroitin sulfate-rich matrices and synthetic scaffolds are needed. We describe the use of both chondroitin sulfate and a novel chondroitin sulfate-binding peptide to control the release of nerve growth factor. Interestingly, the novel chondroitin sulfate-binding peptide enhances the controlled release properties of the chondroitin sulfate gels. While introduction of chondroitin sulfate into a scaffold inhibits primary cortical outgrowth, the combination of chondroitin sulfate, chondroitin sulfate-binding peptide and nerve growth factor promotes primary cortical neurite outgrowth in chondroitin sulfate gels.

In Vivo Assessment of Neurodegeneration in Type C Niemann-Pick Disease by IDEAL-IQ.

Science.gov (United States)

Guo, Ruo-Mi; Li, Qing-Ling; Luo, Zhong-Xing; Tang, Wen; Jiao, Ju; Wang, Jin; Kang, Zhuang; Chen, Shao-Qiong; Zhang, Yong

2018-01-01

To noninvasively assess the neurodegenerative changes in the brain of patients with Niemann-Pick type C (NPC) disease by measuring the lesion tissue with the iterative decomposition of water and fat with echo asymmetry and least square estimation-iron quantification (IDEAL-IQ). Routine brain MRI, IDEAL-IQ and 1 H-proton magnetic resonance spectroscopy ( 1 H-MRS, served as control) were performed on 12 patients with type C Niemann-Pick disease (4 males and 8 females; age range, 15-61 years; mean age, 36 years) and 20 healthy subjects (10 males and 10 females; age range, 20-65 years; mean age, 38 years). The regions with lesion and the normal appearing regions (NARs) of patients were measured and analyzed based on the fat/water signal intensity on IDEAL-IQ and the lipid peak on 1 H-MRS. Niemann-Pick type C patients showed a higher fat/water signal intensity ratio with IDEAL-IQ on T2 hyperintensity lesions and NARs (3.7-4.9%, p IQ instead of 1 H-MRS. The findings of this study suggested that IDEAL-IQ may be useful as a noninvasive and objective method in the evaluation of patients with NPC; additionally, IDEAL-IQ can be used to quantitatively measure the brain parenchymal adipose content and monitor patient follow-up after treatment of NPC.

Immunohistochemical localization of chondroitin sulfate, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, entactin, and laminin in basement membranes of postnatal developing and adult rat lungs

DEFF Research Database (Denmark)

Sannes, P L; Burch, K K; Khosla, J

1993-01-01

Histologic preparations of lungs from 1-, 5-, 10-, 18-, and 25-day-old postnatal and adult rats were examined immunohistochemically with antibodies specific against chondroitin sulfate (CS), basement membrane chondroitin sulfate proteoglycan (BM-CSPG), heparan sulfate proteoglycan (HSPG), entactin...

A novel chondroitin sulfate hydrogel for nerve repair

Science.gov (United States)

Conovaloff, Aaron William

Brachial plexus injuries affect numerous patients every year, with very debilitating results. The majority of these cases are very severe, and involve damage to the nerve roots. To date, repair strategies for these injuries address only gross tissue damage, but do not supply cells with adequate regeneration signals. As a result, functional recovery is often severely lacking. Therefore, a chondroitin sulfate hydrogel that delivers neurotrophic signals to damaged neurons is proposed as a scaffold to support nerve root regeneration. Capillary electrophoresis studies revealed that chondroitin sulfate can physically bind with a variety of neurotrophic factors, and cultures of chick dorsal root ganglia demonstrated robust neurite outgrowth in chondroitin sulfate hydrogels. Outgrowth in chondroitin sulfate gels was greater than that observed in control gels of hyaluronic acid. Furthermore, the chondroitin sulfate hydrogel's binding activity with nerve growth factor could be enhanced by incorporation of a synthetic bioactive peptide, as revealed by fluorescence recovery after photobleaching. This enhanced binding was observed only in chondroitin sulfate gels, and not in hyaluronic acid control gels. This enhanced binding activity resulted in enhanced dorsal root ganglion neurite outgrowth in chondroitin sulfate gels. Finally, the growth of regenerating dorsal root ganglia in these gels was imaged using label-free coherent anti-Stokes scattering microscopy. This technique generated detailed, high-quality images of live dorsal root ganglion neurites, which were comparable to fixed, F-actin-stained samples. Taken together, these results demonstrate the viability of this chondroitin sulfate hydrogel to serve as an effective implantable scaffold to aid in nerve root regeneration.

A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation

Science.gov (United States)

Sattelle, Benedict M.; Shakeri, Javad; Roberts, Ian S.; Almond, Andrew

2010-01-01

The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-strucutre at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without 15N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the β(1→3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the β(1→3) ψ dihedral (as previously predicted by simulation), while the β(1→4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties. PMID:20022001

Chondroitin 4-O-Sulfotransferase Is Indispensable for Sulfation of Chondroitin and Plays an Important Role in Maintaining Normal Life Span and Oxidative Stress Responses in Nematodes*

Science.gov (United States)

Izumikawa, Tomomi; Dejima, Katsufumi; Watamoto, Yukiko; Nomura, Kazuko H.; Kanaki, Nanako; Rikitake, Marika; Tou, Mai; Murata, Daisuke; Yanagita, Eri; Kano, Ai; Mitani, Shohei; Nomura, Kazuya; Kitagawa, Hiroshi

2016-01-01

Chondroitin sulfate (CS)/chondroitin (Chn) chains are indispensable for embryonic cell division and cytokinesis in the early developmental stages in Caenorhabditis elegans and mice, whereas heparan sulfate (HS) is essential for axon guidance during nervous system development. These data indicate that the fundamental functions of CS and HS are conserved from worms to mammals and that the function of CS/Chn differs from that of HS. Although previous studies have shown that C. elegans produces HS and non-sulfated Chn, whether the organism produces CS remains unclear. Here, we demonstrate that C. elegans produces a small amount of 4-O-sulfated Chn and report the identification of C41C4.1, an orthologue of the human chondroitin 4-O-sulfotransferase gene. Loss of C41C4.1 in C. elegans resulted in a decline in 4-O-sulfation of CS and an increase in the number of sulfated units in HS. C41C4.1 deletion mutants exhibited reduced survival rates after synchronization with sodium hypochlorite. Collectively, these results show for the first time that CS glycans are present in C. elegans and that the Chn 4-O-sulfotransferase responsible for the sulfation plays an important role in protecting nematodes from oxidative stress. PMID:27645998

A potential role for chondroitin sulfate/dermatan sulfate in arm regeneration in Amphiura filiformis.

Science.gov (United States)

Ramachandra, Rashmi; Namburi, Ramesh B; Dupont, Sam T; Ortega-Martinez, Olga; van Kuppevelt, Toin H; Lindahl, Ulf; Spillmann, Dorothe

2017-05-01

Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar Amphiura filiformis, with high proportions of di- and tri-O-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during A. filiformis arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-O-sulfated disaccharides. Conversely, treatment of A. filiformis with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1-2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-O-sulfotransferase 2 and dermatan-4-O-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the A. filiformis orthologue of bone morphogenetic factors, AfBMP2/4. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-O-sulfotransferase 2, with a corresponding increase in AfBMP2/4 during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for A. filiformis arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chondroitin sulfate synthase-2 is necessary for chain extension of chondroitin sulfate but not critical for skeletal development.

Science.gov (United States)

Ogawa, Hiroyasu; Hatano, Sonoko; Sugiura, Nobuo; Nagai, Naoko; Sato, Takashi; Shimizu, Katsuji; Kimata, Koji; Narimatsu, Hisashi; Watanabe, Hideto

2012-01-01

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. Based on its structural diversity in chain length and sulfation patterns, CS provides specific biological functions in cell adhesion, morphogenesis, neural network formation, and cell division. To date, six glycosyltransferases are known to be involved in the biosynthesis of chondroitin saccharide chains, and a hetero-oligomer complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is known to have the strongest polymerizing activity. Here, we generated and analyzed CSS2(-/-) mice. Although they were viable and fertile, exhibiting no overt morphological abnormalities or osteoarthritis, their cartilage contained CS chains with a shorter length and at a similar number to wild type. Further analysis using CSS2(-/-) chondrocyte culture systems, together with siRNA of CSS1, revealed the presence of two CS chain species in length, suggesting two steps of CS chain polymerization; i.e., elongation from the linkage region up to Mr ∼10,000, and further extension. There, CSS2 mainly participated in the extension, whereas CSS1 participated in both the extension and the initiation. Our study demonstrates the distinct function of CSS1 and CSS2, providing a clue in the elucidation of the mechanism of CS biosynthesis.

Development of affinity-based delivery of NGF from a chondroitin sulfate biomaterial

OpenAIRE

Butterfield, Karen Chao; Conovaloff, Aaron W.; Panitch, Alyssa

2011-01-01

Chondroitin sulfate is a major component of the extracellular matrix in both the central and peripheral nervous systems. Chondroitin sulfate is upregulated at injury, thus methods to promote neurite extension through chondroitin sulfate-rich matrices and synthetic scaffolds are needed. We describe the use of both chondroitin sulfate and a novel chondroitin sulfate-binding peptide to control the release of nerve growth factor. Interestingly, the novel chondroitin sulfate-binding peptide enhanc...

Chondroitin 4-O-Sulfotransferase Is Indispensable for Sulfation of Chondroitin and Plays an Important Role in Maintaining Normal Life Span and Oxidative Stress Responses in Nematodes.

Science.gov (United States)

Izumikawa, Tomomi; Dejima, Katsufumi; Watamoto, Yukiko; Nomura, Kazuko H; Kanaki, Nanako; Rikitake, Marika; Tou, Mai; Murata, Daisuke; Yanagita, Eri; Kano, Ai; Mitani, Shohei; Nomura, Kazuya; Kitagawa, Hiroshi

2016-10-28

Chondroitin sulfate (CS)/chondroitin (Chn) chains are indispensable for embryonic cell division and cytokinesis in the early developmental stages in Caenorhabditis elegans and mice, whereas heparan sulfate (HS) is essential for axon guidance during nervous system development. These data indicate that the fundamental functions of CS and HS are conserved from worms to mammals and that the function of CS/Chn differs from that of HS. Although previous studies have shown that C. elegans produces HS and non-sulfated Chn, whether the organism produces CS remains unclear. Here, we demonstrate that C. elegans produces a small amount of 4-O-sulfated Chn and report the identification of C41C4.1, an orthologue of the human chondroitin 4-O-sulfotransferase gene. Loss of C41C4.1 in C. elegans resulted in a decline in 4-O-sulfation of CS and an increase in the number of sulfated units in HS. C41C4.1 deletion mutants exhibited reduced survival rates after synchronization with sodium hypochlorite. Collectively, these results show for the first time that CS glycans are present in C. elegans and that the Chn 4-O-sulfotransferase responsible for the sulfation plays an important role in protecting nematodes from oxidative stress. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

DEFF Research Database (Denmark)

McCarthy, K J; Accavitti, M A; Couchman, J R

1989-01-01

with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were...... (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core...... sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component....

Immune dysfunction in Niemann?Pick disease type C

OpenAIRE

Platt, Nick; Speak, Annelise O.; Colaco, Alexandria; Gray, James; Smith, David A.; Williams, Ian M.; Wallom, Kerri?Lee; Platt, Frances M.

2015-01-01

Abstract Lysosomal storage diseases are inherited monogenic disorders in which lysosome function is compromised. Although individually very rare, they occur at a collective frequency of approximately one in five thousand live births and usually have catastrophic consequences for health. The lysosomal storage diseases Niemann?Pick disease type C (NPC) is caused by mutations predominantly in the lysosomal integral membrane protein NPC1 and clinically presents as a progressive neurodegenerative ...

Two fucosylated chondroitin sulfates from the sea cucumber Eupentacta fraudatrix.

Science.gov (United States)

Ustyuzhanina, Nadezhda E; Bilan, Maria I; Dmitrenok, Andrey S; Nifantiev, Nikolay E; Usov, Anatolii I

2017-05-15

Two fucosylated chondroitin sulfates EF1 and EF2 were isolated from the sea cucumber Eupentacta fraudatrix. Separation of the polysaccharides was performed using anion-exchange chromatography on DEAE-Sephacel by elution of 0.75M and 1.0M NaCl solutions. The structures of biopolymers were determined by chemical and NMR spectroscopic methods. The backbone of EF1 was found to be composed of chondroitin sulfate A and E units in a ratio of about 1:1. The core of EF2 along with chondroitin sulfate A and E fragments contained unusual disaccharide repeating units →4)-β-d-GlcpA2S3S-(1→3)-β-d-GalpNAc6S-(1→. The main type of branches in both polysaccharides was α-l-Fucp3S4S unit attached to O-3 of GlcA residues. Another type of branches was found to be the disaccharide fragment α-l-Fucp-(1→2)-α-l-Fucp3S4S-(1→ linked to O-3 of GlcA. The presence of structurally different fucosylated chondroitin sulfates in one species of sea cucumber is rather unusual and has not been described previously. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dietary cholesterol induces trafficking of intestinal Niemann-Pick Type C1 Like 1 from the brush border to endosomes

DEFF Research Database (Denmark)

Skov, Marianne; Tønnesen, Carina K; Hansen, Gert H

2011-01-01

The transmembrane protein Niemann-Pick C1 Like 1 (NPC1L1) belongs to the Niemann-Pick C1 (NPC1) family of cholesterol transporters and is mainly expressed in the liver and the small intestine. NPC1L1 is believed to be the main transporter responsible for the absorption of dietary cholesterol. Lik...

Photorefractive keratectomy: measuring the matrix metalloproteinase activity and chondroitin sulfate concentration in tear fluid

Directory of Open Access Journals (Sweden)

Tetsuya Mutoh

2010-09-01

Full Text Available Tetsuya Mutoh, Masaya Nishio, Yukihiro Matsumoto, Kiyomi Arai, Makoto ChikudaDepartment of Ophthalmology, Dokkyo Medical University Koshigaya Hospital, Saitama, JapanAbstract: We herein report the case of a 20-year-old man who underwent a photorefractive keratectomy (PRK. We measured matrix metalloproteinase-9 (MMP-9 activity and chondroitin 4 sulfate and chondroitin 6 sulfate concentrations in tear fluid. Tear fluid was collected preoperatively via microcapillary tube, and was collected postoperatively on the first and fourth days, and after one week, one month, three months, and six months. Samples were formulated by dilution with 200 µL of saline. MMP-9 activity was analyzed by an enzyme immunocapture activity assay, and the concentrations of chondroitin sulfate were analyzed by enzyme-linked immunosorbent assay. No complications were observed after surgery, except for a minimal subepithelial haze. Although MMP-9 activity changed on the fourth postoperative day, the activity changed only minimally at this time. Chondroitin 4 sulfate concentrations in tear fluid increased dramatically from one week to one month, decreased transiently at three months, and increased by six months. The chondroitin 6 sulfate concentration did not normalize within one week, and decreased from one week to three months compared with the preoperative score, and was close to the preoperative score at six months. We conclude that corneal wound healing was still incomplete six months after PRK, and chondroitin 4 sulfate appears to be critical in this process.Keywords: matrix metalloproteinase, chondroitin sulfate, human tear fluid, photorefractive keratectomy, corneal wound healing

[Analysis of chondroitin sulfate content of Cervi Cornu Pantotrichum with different processing methods and different parts].

Science.gov (United States)

Gong, Rui-Ze; Wang, Yan-Hua; Sun, Yin-Shi

2018-02-01

The differences and the variations of chondroitin sulfate content in different parts of Cervi Cornu Pantotrichum(CCP) with different processing methods were investigated. The chondroitin sulfate from velvet was extracted by dilute alkali-concentrated salt method. Next， the chondroitin sulfate was digested by chondroitinase ABC.The contents of total chondroitin sulfate and chondroitin sulfate A， B and C in the samples were determined by high performance liquid chromatography(HPLC).The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with freeze-drying processing is 14.13，11.99，1.74，0.32 g·kg⁻¹， respectively. The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with boiling processing is 10.71，8.97，2.21，1.40 g·kg⁻¹， respectively. The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP without blood is 12.47，9.47，2.64，0.07 g·kg⁻¹， respectively. And the content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with blood is 8.22，4.39，0.87，0.28 g·kg⁻¹ respectively. The results indicated that the chondroitin sulfate content in different processing methods was significantly different.The content of chondroitin sulfate in CCP with freeze-drying is higher than that in CCP with boiling processing.The content of chondroitin sulfate in CCP without blood is higher than that in CCP with blood. The chondroitin sulfate content in differerent paris of the velvet with the same processing methods was arranged from high to low as： wax slices， powder， gauze slices， bone slices. Copyright© by the Chinese Pharmaceutical Association.

Inhibitory effect of chondroitin sulfate oligosaccharides on bovine testicular hyaluronidase.

Science.gov (United States)

Kakizaki, Ikuko; Koizumi, Hideyo; Chen, Fengchao; Endo, Masahiko

2015-05-05

Hyaluronan and chondroitin sulfates are prominent components of the extracellular matrices of animal tissues; however, their functions in relation to their oligosaccharide structures have not yet been fully elucidated. The oligosaccharides of hyaluronan and chondroitin sulfate were prepared and used to investigate their effects on the hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase when hyaluronan was used as a substrate. Hydrolysis and transglycosylation activities were assessed in independent reaction systems by analyzing the products by HPLC. The hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase were dose-dependently inhibited by chondroitin sulfate oligosaccharides, but not by hyaluronan or chondroitin oligosaccharides. A kinetic analysis of the hydrolysis reaction using hyaluronan octasaccharide revealed that the inhibition mode by chondroitin sulfate oligosaccharides was competitive. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hexagonal-shaped chondroitin sulfate self-assemblies have exalted anti-HSV-2 activity.

Science.gov (United States)

Galus, Aurélia; Mallet, Jean-Maurice; Lembo, David; Cagno, Valeria; Djabourov, Madeleine; Lortat-Jacob, Hugues; Bouchemal, Kawthar

2016-01-20

The initial step in mucosal infection by the herpes simplex virus type 2 (HSV-2) requires its binding to certain glycosaminoglycans naturally present on host cell membranes. We took advantage of this interaction to design biomimetic supramolecular hexagonal-shaped nanoassemblies composed of chondroitin sulfate having exalted anti-HSV-2 activity in comparison with native chondroitin sulfate. Nanoassemblies were formed by mixing hydrophobically-modified chondroitin sulfate with α-cyclodextrin in water. Optimization of alkyl chain length grafted on chondroitin sulfate and the ratio between hydrophobically-modified chondroitin sulfate and α-cyclodextrin showed that more cohesive and well-structured nanoassemblies were obtained using higher α-cyclodextrin concentration and longer alkyl chain lengths. A structure-activity relationship was found between anti-HSV-2 activity and the amphiphilic nature of hydrophobically-modified chondroitin sulfate. Also, antiviral activity of hexagonal nanoassemblies against HSV-2 was further improved in comparison with hydrophobically-modified chondroitin sulfate. This work suggests a new biomimetic formulation approach that can be extended to other heparan-sulfate-dependent viruses. Copyright © 2015 Elsevier Ltd. All rights reserved.

Galactose 6-sulfate sulfatase activity in Morquio syndrome

International Nuclear Information System (INIS)

Yutaka, T.; Okada, S.; Kato, T.; Inui, K.; Yabuuhi, H.

1982-01-01

The authors have prepared a new substrate, o-β-D-sulfo-galactosyl-(1-4)-β-D-6-sulfo-2-acetamido-2-deoxyglucosyl-(1-4)-D-[1- 3 H]galactitol, from shark cartilage keratan sulfate, for the assay of galactose 6-sulfate sulfatase activity. Using this substrate, they found there was a striking deficiency of galactose 6-sulfate sulfatase activity, in addition to the known deficiency of N-acetylgalactosamine 6-sulfate sulfatase, in the cultured skin fibroblasts of patients with Morquio syndrome. Their results could be explained by the hypothesis that accumulation of keratan sulfate and chondroitin 6-sulfate in Morquio syndrome is due to a deficiency of galactose 6-sulfate sulfatase and N-acetylgalactosamine 6-sulfate sulfatase activity, which are necessary for the degradation of these two mucopolysaccharides. (Auth.)

Galactose 6-sulfate sulfatase activity in Morquio syndrome

Energy Technology Data Exchange (ETDEWEB)

Yutaka, T.; Okada, S.; Kato, T.; Inui, K.; Yabuuhi, H. (Osaka Univ. (Japan). Faculty of Medicine)

1982-07-01

The authors have prepared a new substrate, o-..beta..-D-sulfo-galactosyl-(1-4)-..beta..-D-6-sulfo-2-acetamido-2-deoxyglucosyl-(1-4)-D-(1-/sup 3/H)galactitol, from shark cartilage keratan sulfate, for the assay of galactose 6-sulfate sulfatase activity. Using this substrate, they found there was a striking deficiency of galactose 6-sulfate sulfatase activity, in addition to the known deficiency of N-acetylgalactosamine 6-sulfate sulfatase, in the cultured skin fibroblasts of patients with Morquio syndrome. Their results could be explained by the hypothesis that accumulation of keratan sulfate and chondroitin 6-sulfate in Morquio syndrome is due to a deficiency of galactose 6-sulfate sulfatase and N-acetylgalactosamine 6-sulfate sulfatase activity, which are necessary for the degradation of these two mucopolysaccharides.

Characterization of a chondroitin sulfate hydrogel for nerve root regeneration

Science.gov (United States)

Conovaloff, Aaron; Panitch, Alyssa

2011-10-01

Brachial plexus injury is a serious medical problem that affects many patients annually, with most cases involving damage to the nerve roots. Therefore, a chondroitin sulfate hydrogel was designed to both serve as a scaffold for regenerating root neurons and deliver neurotrophic signals. Capillary electrophoresis showed that chondroitin sulfate has a dissociation constant in the micromolar range with several common neurotrophins, and this was determined to be approximately tenfold stronger than with heparin. It was also revealed that nerve growth factor exhibits a slightly stronger affinity for hyaluronic acid than for chondroitin sulfate. However, E8 chick dorsal root ganglia cultured in the presence of nerve growth factor revealed that ganglia cultured in chondroitin sulfate scaffolds showed more robust growth than those cultured in control gels of hyaluronic acid. It is hypothesized that, despite the stronger affinity of nerve growth factor for hyaluronic acid, chondroitin sulfate serves as a better scaffold for neurite outgrowth, possibly due to inhibition of growth by hyaluronic acid chains.

Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes

International Nuclear Information System (INIS)

Uhlin-Hansen, L.; Eskeland, T.; Kolset, S.O.

1989-01-01

Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of [35S]chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the [35S]CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains

Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

International Nuclear Information System (INIS)

Ruffell, Brian; Johnson, Pauline

2005-01-01

CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding

The combined therapy with chondroitin sulfate plus glucosamine sulfate or chondroitin sulfate plus glucosamine hydrochloride does not improve joint damage in an experimental model of knee osteoarthritis in rabbits.

Science.gov (United States)

Roman-Blas, Jorge A; Mediero, Aránzazu; Tardío, Lidia; Portal-Nuñez, Sergio; Gratal, Paula; Herrero-Beaumont, Gabriel; Largo, Raquel

2017-01-05

Osteoarthritis is the most common chronic joint disorder especially during aging. Although with controversies, glucosamine, both in its forms of sulfate and hydrochloride, and chondroitin sulfate are commonly employed to treat osteoarthritis. Due to the modest improve in the symptoms observed in patients treated with these drugs alone, a formulation combining both agents has been considered. The discrepant results achieved for pain control or structural improvement in osteoarthritis patients has been attributed to the quality of chemical formulations or different bias in clinical studies. The current study has been designed to test the effects of two different combined formulations with adequate pharmaceutical grade of these drugs in osteoarthritic joints, and to explore the underlying mechanisms modulated by both formulations in different osteoarthritis target tissues. Knee osteoarthritis was surgically induced in experimental rabbits. Some animals received the combined therapy (CT)1, (chondroitin sulfate 1200mg/day + glucosamine sulfate 1500mg/day), or the CT2 ((chondroitin sulfate 1200mg/day + glucosamine hydrochloride 1500mg/day). Neither CT1 nor CT2 significantly modified the cartilage damage or the synovial inflammation observed in osteoarthritic animals. Treatments were also unable to modify the presence of pro-inflammatory mediators, and the synthesis of metalloproteinases in the cartilage or in the synovium of osteoarthritic animals. Combined therapies did not modify the decrease in the subchondral bone mineral density observed in osteoarthritic rabbits. Therapies of chondroitin sulfate plus glucosamine sulfate or chondroitin sulfate plus glucosamine hydrochloride failed to improve structural damage or to ameliorate the inflammatory profile of joint tissues during experimental osteoarthritis. Published by Elsevier B.V.

Acute Exacerbations of COPD Are Associated With Increased Expression of Heparan Sulfate and Chondroitin Sulfate in BAL.

Science.gov (United States)

Papakonstantinou, Eleni; Klagas, Ioannis; Roth, Michael; Tamm, Michael; Stolz, Daiana

2016-03-01

Acute exacerbations of COPD (AECOPDs) are associated with accelerated aggravation of clinical symptoms and deterioration of pulmonary function. The mechanisms by which exacerbations may contribute to airway remodeling and declined lung function are poorly understood. We investigated whether AECOPDs are associated with differential expression of glycosaminoglycans in BAL in a cohort of 97 patients with COPD. Patients with COPD with either stable disease (n = 53) or AECOPD (n = 44) and undergoing diagnostic bronchoscopy were matched for demographics and lung function parameters. Levels of heparan sulfate, chondroitin sulfate, dermatan sulfate, and matrix metalloproteinases (MMPs) in BAL were measured by enzyme-linked immunosorbent assay. Heparan sulfate and chondroitin sulfate were significantly increased in BAL of patients during exacerbations. Levels of heparan sulfate were higher in the BAL of patients with microbial infections. Chondroitin sulfate was negatively correlated with FEV1 % predicted but not with diffusing capacity of lung for carbon monoxide % predicted, indicating that chondroitin sulfate is associated with airway remodeling, leading to obstruction rather than to emphysema. Furthermore, heparan sulfate and chondroitin sulfate were significantly correlated with MMP-9, MMP-2, and MMP-12 in BAL, indicating that they were cleaved from their respective proteoglycans by MMPs and subsequently washed out in BAL. During AECOPD, there is increased expression of heparan sulfate and chondroitin sulfate in BAL. These molecules are significantly correlated with MMPs in BAL, indicating that they may be associated with airway remodeling and may lead to lung function decline during exacerbations of COPD. Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

Inter vs. intraglycosidic acetal linkages control sulfation pattern in semi-synthetic chondroitin sulfate.

Science.gov (United States)

Laezza, Antonio; De Castro, Cristina; Parrilli, Michelangelo; Bedini, Emiliano

2014-11-04

Microbial-sourced unsulfated chondroitin could be converted into chondroitin sulfate (CS) polysaccharide by a multi-step strategy relying upon benzylidenation and acetylation reactions as key-steps for its regioselective protection. By conducting the two reactions one- or two-pots, CSs with different sulfation patterns could be obtained at the end of the semi-synthesis. In particular, a CS polysaccharide possessing sulfate groups randomly distributed between positions 4 and 6 of N-acetyl-galactosamine (GalNAc) units could be obtained through the two-pots route, whereas the one-pot pathway allowed an additional sulfation at position 3 of some glucuronic acid (GlcA) units. This difference was ascribed to the stabilization of a labile interglycosidic benzylidene acetal involving positions O-3 and O-6 of some GlcA and GalNAc, respectively, when the benzylidene-acetylation reactions were conducted in a one-pot fashion. Isolation and characterization of a polysaccharide intermediate showing interglycosidic acetal moieties was accomplished. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enfermedad de Niemann Pick tipo-A. Presentación de 12 casos

Directory of Open Access Journals (Sweden)

Jorge Zarco-Román

2017-05-01

Full Text Available INTRODUCCIÓN: la enfermedad de Niemann Pick tipo A es una enfermedad hereditaria de depósito lisosomal, debida a un déficit de la enzima esfingomielinasa ácida, lo que da lugar a la acumulación de esfingomielina en las células del sistema reticuloendotelial y nervioso central. OBJETIVO: se describe el curso de la enfermedad de Niemann Pick tipo A en 12 pacientes: tres niños y nueve niñas, con edades entre 3 a 33 meses. MATERIAL Y MÉTODOS: estudio retrospectivo efectuado con base en la información de los expedientes de 12 pacientes, evaluados mediante un examen físico completo, oftalmológico, análisis de laboratorio, ultrasonido de abdomen, tomografía axial computarizada o resonancia magnética nuclear cerebral, radiografías de tórax, ecocardiograma, electroencefalograma, potenciales evocados auditivos y visuales, serie esofagogastroduodenal y serie ósea. Se obtuvo información sobre enfermedades intercurrentes y causas de muerte. RESULTADOS: la sintomatología comenzó en promedio a los 5.9 meses (2 a 16 meses y el diagnóstico definitivo se realizó a la edad de los 15.6 meses (3 a 33 meses a través del cuadro clínico sugestivo, con presencia de células espumosas en el aspirado de médula ósea 12/12 (100% y deficiencia de la enzima esfingomielinasa ácida en 12/12 pacientes (100%; la biopsia hepática fue sugestiva de enfermedad por atesoramiento en 5/12 (41.6%. Las manifestaciones clínicas por orden de frecuencia fueron: hepatoesplenomegalia y afección neurológica en 12/12 (100%, alteración pulmonar 11/12 (91.6%, hematológica 9/12 (80%, oftalmológica con mancha rojo cereza 9/12 (75%, ósea 6/12 (50%, y cardiaca 4/12 (33.3%. Únicamente hubo antecedente de consanguinidad en 4 familias (33.3%. CONCLUSIONES: el curso clínico de la enfermedad de Niemann Pick tipo A es muy similar entre los pacientes afectados, el primer dato clínico detectado es la hepatoesplenomegalia. Los pacientes están asintomáticos los primeros

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species.

OpenAIRE

Lipeski, L; Guthrie, E P; O'Brien, M; Kotarski, S F; Salyers, A A

1986-01-01

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetai...

Chondroitin sulfates do not impede axonal regeneration in goldfish spinal cord.

Science.gov (United States)

Takeda, Akihito; Okada, Soichiro; Funakoshi, Kengo

2017-10-15

Chondroitin sulfate proteoglycans produced in glial scar tissue are a major inhibitory factor for axonal regeneration after central nervous system injury in mammals. The inhibition is largely due to chondroitin sulfates, whose effects differ according to the sulfation pattern. In contrast to mammals, fish nerves spontaneously regenerate beyond the scar tissue after spinal cord injury, although the mechanisms that allow for axons to pass through the scar are unclear. Here, we used immunohistochemistry to examine the expression of two chondroitin sulfates with different sulfation variants at the lesion site in goldfish spinal cord. The intact spinal cord was immunoreactive for both chondroitin sulfate-A (CS-A) and chondroitin sulfate-C (CS-C), and CS-A immunoreactivity overlapped extensively with glial processes positive for glial fibrillary acidic protein. At 1week after inducing the spinal lesion, CS-A immunoreactivity was observed in the cell bodies and extracellular matrix, as well as in glial processes surrounding the lesion center. At 2weeks after the spinal lesion, regenerating axons entering the lesion center overtook the CS-A abundant area. In contrast, at 1week after lesion induction, CS-C immunoreactivity was significantly decreased, and at 2weeks after lesion induction, CS-C immunoreactivity was observed along the regenerating axons entering the lesion center. The present findings suggest that after spinal cord injury in goldfish, chondroitin sulfate proteoglycans are deposited in the extracellular matrix at the lesion site but do not form an impenetrable barrier to the growth of regenerating axons. Copyright © 2017 Elsevier B.V. All rights reserved.

Discrepancies in composition and biological effects of different formulations of chondroitin sulfate.

Science.gov (United States)

Martel-Pelletier, Johanne; Farran, Aina; Montell, Eulàlia; Vergés, Josep; Pelletier, Jean-Pierre

2015-03-06

Osteoarthritis is a common, progressive joint disease, and treatments generally aim for symptomatic improvement. However, SYmptomatic Slow-Acting Drugs in Osteoarthritis (SYSADOAs) not only reduce joint pain, but slow structural disease progression. One such agent is chondroitin sulfate-a complex, heterogeneous polysaccharide. It is extracted from various animal cartilages, thus has a wide range of molecular weights and different amounts and patterns of sulfation. Chondroitin sulfate has an excellent safety profile, and although various meta-analyses have concluded that it has a beneficial effect on symptoms and structure, others have concluded little or no benefit. This may be due, at least partly, to variations in the quality of the chondroitin sulfate used for a particular study. Chondroitin sulfate is available as pharmaceutical- and nutraceutical-grade products, and the latter have great variations in preparation, composition, purity and effects. Moreover, some products contain a negligible amount of chondroitin sulfate and among samples with reasonable amounts, in vitro testing showed widely varying effects. Of importance, although some showed anti-inflammatory effects, others demonstrated weak effects, and some instances were even pro-inflammatory. This could be related to contaminants, which depend on the origin, production and purification process. It is therefore vitally important that only pharmaceutical-grade chondroitin sulfate be used for treating osteoarthritis patients.

A case of variant biochemical phenotype of Niemann-Pick disease type C accompanying savant syndrome.

Science.gov (United States)

Hamatani, Mio; Jingami, Naoto; Uemura, Kengo; Nakasone, Naoe; Kinoshita, Hisanori; Yamakado, Hodaka; Ninomiya, Haruaki; Takahashi, Ryosuke

2016-06-22

A 40-year-old man was referred to our hospital because of vertical supranuclear gaze palsy, frequent sudden loss of muscle tonus and ataxia for several years. He had a history of prolonged neonatal jaundice. He was given a diagnosis of autism in his childhood, followed by a diagnosis of schizophrenia in his teenage. He also developed a savant skill of calendar calculating. (123)I-IMP-SPECT showed decreased cerebral blood flow in the left frontotemporal lobe as often seen in savant syndrome. Although genetic analysis of NPC1 and NPC2 revealed no pathogenic mutation, filipin staining of cultured fibroblasts from his biopsied skin revealed a certain amount of intracellular cholesterol storage pattern, indicating a variant biochemical phenotype of Niemann-Pick disease type C (NPC). The diagnosis of adulthood onset NPC is difficult and challenging, especially for neurologists, because the symptoms and signs are not as clear as those in the classical childhood onset NPC and this subtype is not yet widely known. However, the diagnosis can be made by a combination of filipin staining of fibroblast and/or gene analysis. As a disease-specific therapy for NPC has been approved in Japan, the diagnosis of NPC is of significance.

Symptom and structure modification in osteoarthritis with pharmaceutical-grade chondroitin sulfate: what's the evidence?

Science.gov (United States)

Hochberg, M; Chevalier, X; Henrotin, Y; Hunter, D J; Uebelhart, D

2013-03-01

Osteoarthritis is a chronic disease characterized by irreversible damage to joint structures, including loss of articular cartilage, osteophyte formation, alterations in the subchondral bone and synovial inflammation. It has been shown that chondroitin sulfate interferes with the progression of structural changes in joint tissues and is used in the management of patients with osteoarthritis. This review summarizes data from relevant reports describing the mechanisms of action of chondroitin sulfate that may explain the beneficial effects of the drug and examines the evidence for clinical efficacy of oral chondroitin sulfate in osteoarthritis. Data included in the review were derived from a literature search in PubMed. Literature searches were performed in PubMed using the search terms 'chondroitin sulfate', 'pharmaceutical-grade', 'osteoarthritis', 'randomized clinical trials', 'humans'. The MEDLINE database was searched from January 1996 through August 2012 for all randomized controlled trials, meta-analyses, systematic reviews, and review articles of chondroitin sulfate in osteoarthritis. Chondroitin sulfate exerts in vitro a beneficial effect on the metabolism of different cell lines: chondrocytes, synoviocytes and cells from subchondral bone, all involved in osteoarthritis. It increases type II collagen and proteoglycan synthesis in human articular chondrocytes and is able to reduce the production of some pro-inflammatory factors and proteases, to reduce the cellular death process, and improve the anabolic/catabolic balance of the extracellular cartilage matrix (ECM). Clinical trials have reported a beneficial effect of chondroitin sulfate on pain and function. The structure-modifying effects of chondroitin sulfate have been reported and analyzed in recent meta-analyses. The results in knee osteoarthritis demonstrate a small but significant reduction in the rate of decline in joint space width. Because chondroitin sulfate quality of several nutraceuticals has

Biological functions of iduronic acid in chondroitin/dermatan sulfate

OpenAIRE

Thelin, Martin A; Bartolini, Barbara; Axelsson, Jakob; Gustafsson, Renata; Tykesson, Emil; Pera, Edgar; Oldberg, ?ke; Maccarana, Marco; Malmstrom, Anders

2013-01-01

The presence of iduronic acid in chondroitin/dermatan sulfate changes the properties of the polysaccharides because it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/dermatan sulfate influences multiple cellular properties, such as migration, proliferation, differentiation, angiogenesis and the regulation of cytokine/growth factor activities. Under pathological conditions such as wound healing, inflammation and cancer, iduronic acid has diverse...

Basement membrane chondroitin sulfate proteoglycan alterations in a rat model of polycystic kidney disease

DEFF Research Database (Denmark)

Ehara, T; Carone, F A; McCarthy, K J

1994-01-01

of distal tubules and collecting ducts was observed by 4 days with phenol II treatment, but the morphology returned to normal after 7 days of subsequent normal diet. Staining of tissue sections with two mouse monoclonal antibodies to a recently described basement membrane chondroitin sulfate proteoglycan...... to chondroitin sulfate chains confirmed these changes in cystic tubule basement membranes. During the recovery stage, interstitial chondroitin sulfate (representing a CSPG other than BM-CSPG) was greatly increased around these tubules, along with the glycoprotein fibronectin. Staining with antibody to a basement...... membrane heparan sulfate proteoglycan core protein related to perlecan did not diminish but rather stained affected tubules intensely, whereas laminin, on the other hand, was apparently diminished in the basement membranes of the cystic tubules. Type IV collagen staining did not change through disease...

Development of a Suspicion Index to aid diagnosis of Niemann-Pick disease type C

NARCIS (Netherlands)

Wijburg, F. A.; Sedel, F.; Pineda, M.; Hendriksz, C. J.; Fahey, M.; Walterfang, M.; Patterson, M. C.; Wraith, J. E.; Kolb, S. A.

2012-01-01

Objectives: Niemann-Pick disease type C (NP-C) is a rare, autosomal recessive lysosomal lipid storage disorder that is invariably fatal. NP-C diagnosis can be delayed for years due to heterogeneous presentation; adult-onset NP-C can be particularly difficult to diagnose. We developed a Suspicion

The hidden Niemann-Pick type C patient : Clinical niches for a rare inherited metabolic disease

NARCIS (Netherlands)

Hendriksz, Christian J.; Anheim, Mathieu; Bauer, Peter; Bonnot, Olivier; Chakrapani, Anupam; Corvol, Jean-Christophe; de Koning, Tom J.; Degtyareva, Anna; Dionisi-Vici, Carlo; Doss, Sarah; Duning, Thomas; Giunti, Paola; Iodice, Rosa; Johnston, Tracy; Kelly, Dierdre; Kluenemann, Hans-Hermann; Lorenzl, Stefan; Padovani, Alessandro; Pocovi, Miguel; Synofzik, Matthis; Terblanche, Alta; Bergh, Florian Then; Topcu, Meral; Tranchant, Christine; Walterfang, Mark; Velten, Christian; Kolb, Stefan A.

2017-01-01

Background: Niemann-Pick disease type C (NP-C) is a rare, inherited neurodegenerative disease of impaired intracellular lipid trafficking. Clinical symptoms are highly heterogeneous, including neurological, visceral, or psychiatric manifestations. The incidence of NP-C is under-estimated due to

Purification, structural characterization and antiproliferative properties of chondroitin sulfate/dermatan sulfate from tunisian fish skins.

Science.gov (United States)

Krichen, Fatma; Volpi, Nicola; Sila, Assaâd; Maccari, Francesca; Mantovani, Veronica; Galeotti, Fabio; Ellouz-Chaabouni, Semia; Bougatef, Ali

2017-02-01

Chondroitin sulfate/dermatan sulfate GAGs were extracted and purified from the skins of grey triggerfish (GTSG) and smooth hound (SHSG). The disaccharide composition produced by chondroitinase ABC treatment showed the presence of nonsulfated disaccharide, monosulfated disaccharides ΔDi6S and ΔDi4S, and disulfated disaccharides in different percentages. In particular, the nonsulfated disaccharide ΔDi0S of GTSG and SHSG were 3.5% and 5.5%, respectively, while monosulfated disaccharides ΔDi6S and ΔDi4S were evaluated to be 18.2%, 59% and 14.6%, 47.0%, respectively. Capillary elecrophoresis analysis of GTSG and SHSG contained 99.2% and 95.4% of chondroitin sulfate/dermatan sulfate, respectively. PAGE analysis showed a GTSG and SHSG having molecular masses with average values of 41.72KDa and 23.8KDa, respectively. HCT116 cell proliferation was inhibited (p<0.05) by 70.6% and 72.65% at 200μg/mL of GTSG and SHSG respectively. Both GTSG and SHSG demonstrated promising antiproliferative potential, which may be used as a novel, effective agent. Copyright © 2016 Elsevier B.V. All rights reserved.

Sulfation pattern of fucose branches affects the anti-hyperlipidemic activities of fucosylated chondroitin sulfate.

Science.gov (United States)

Wu, Nian; Zhang, Yu; Ye, Xingqian; Hu, Yaqin; Ding, Tian; Chen, Shiguo

2016-08-20

Fucosylated chondroitin sulfates (fCSs) are glycosaminoglycans extracted from sea cucumbers, consisting of chondroitin sulfate E (CSE) backbones and sulfated fucose branches. The biological properties of fCSs could be affected by the sulfation pattern of their fucose branches. In the present study, two fCSs were isolated from sea cucumbers Isostichopus badionotus (fCS-Ib) and Pearsonothuria graeffei (fCS-Pg). Their monosaccharide compositions of glucuronic acid (GlcA), N-acetylgalactosamine (GalNAc), fucose (Fuc) and sulfate were at similar molar ratio with 1.0/0.7/0.9/3.1 for fCS-Ib and 1.0/0.8/1.5/2.6 for fCS-Pg. The two fCSs have different sulfation patterns on their fucose branches, fCS-Pg with 3,4-O-disulfation while fCS-Ib with 2,4-O-disulfation. Their antihyperlipidemic effects were compared using a high-fat high-fructose diet (HFFD)-fed C57BL/6J mice model. Both fCS-Ib and fCS-Pg had significant effects on lipid profile improvement, liver protection, blood glucose diminution and hepatic glycogen synthesis. Specifically, fCS-Pg with 3,4-O-disulfation fucose branches was more effective in reduction of blood cholesterol (TC), low density lipoprotein (LDL) and atherogenic index (AI). Our results indicate that both fCSs, especially fCS-Pg, could be used as a potential anti-hyperlipidemic drug. Copyright © 2016. Published by Elsevier Ltd.

Plasma chitotriosidase and CCL18: Early biochemical surrogate markers in type B Niemann-Pick disease

NARCIS (Netherlands)

Brinkman, J.; Wijburg, F. A.; Hollak, C. E.; Groener, J. E.; Verhoek, M.; Scheij, S.; Aten, J.; Boot, R. G.; Aerts, J. M.

2005-01-01

Type B Niemann-Pick disease (NPD) is a nonneuronopathic lysosomal storage disorder which is characterized by accumulation of sphingomyelin-laden macrophages. The availability of plasma markers for storage cells may be of great value in facilitating therapeutic decisions. Given the similarity of the

Oncofetal chondroitin sulfate glycosaminoglycans are key players in integrin signaling and tumor cell motility

DEFF Research Database (Denmark)

Clausen, Thomas Mandel; Bento Ayres Pereira, Marina Maria; Al Nakouzi, Nader

2016-01-01

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2...... revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-β1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes β-1......,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor...

Type a niemann-pick disease. Description of three cases with delayed myelination.

Science.gov (United States)

D'Amico, A; Sibilio, M; Caranci, F; Bartiromo, F; Taurisano, R; Balivo, F; Melis, D; Parenti, G; Cirillo, S; Elefante, R; Brunetti, A

2008-06-03

We describe three patients with type A Niemann-Pick disease (NPD-A). NPD-A is an autosomal recessive neuronal storage disease classified among the sphingolipidoses, characterized by accumulation of sphingomyelin in various tissues and in the brain. Magnetic Resonance imaging (MRI) of our three patients showed a marked delay of myelination with frontal atrophy. Few descriptions of this MRI pattern of delayed myelination have been published to date.

Basement membrane chondroitin sulfate proteoglycans: localization in adult rat tissues

DEFF Research Database (Denmark)

McCarthy, K J; Couchman, J R

1990-01-01

Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production...... and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution...... of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan...

Effects of chondroitin sulfate and glucosamine in adult patients with Kaschin-Beck disease

DEFF Research Database (Denmark)

Zhang, Ya-xu; Dong, Wei; Liu, Hui

2010-01-01

The purpose is to investigate the effects of chondroitin sulfate and glucosamine on adult patients with Kaschin-Beck disease (KBD). A total of 80 patients, aged over 40 years, were randomized into two groups receiving either 1,600 mg oral mixture of chondroitin sulfate and glucosamine or placebo......). But the overall mean change in joint space was significant between the two groups (P chondroitin sulfate and glucosamine might play a protective role in preserving articular cartilage and provide...

Effects of oversulfated and fucosylated chondroitin sulfates on coagulation. Challenges for the study of anticoagulant polysaccharides.

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Fonseca, Roberto J C; Oliveira, Stephan-Nicollas M C G; Pomin, Vitor H; Mecawi, André S; Araujo, Iracema G; Mourão, Paulo A S

2010-05-01

We report the effects of a chemically oversulfated chondroitin sulfate and a naturally fucosylated chondroitin sulfate on the coagulation system. The former has been recently identified as a contaminant of heparin preparations and the latter has been proposed as an alternative anticoagulant. The mechanism of action of these polymers on coagulation is complex and target different components of the coagulation system. They have serpin-independent anticoagulant activity, which preponderates in plasma. They also have serpin-dependent anticoagulant activity but differ significantly in the target coagulation protease and preferential serpin. Their anticoagulant effects differ even more markedly when tested as inhibitors of coagulation proteases using plasma as a source of serpins. It is possible that the difference is due to the high availability of fucosylated chondroitin sulfate whereas oversulfated chondroitin sulfate has strong unspecific binding to plasma protein and low availability for the binding to serpins. When tested using a venous thrombosis experimental model, oversulfated chondroitin sulfate is less potent as an antithrombotic agent than fucosylated chondroitin sulfate. These highly sulfated chondroitin sulfates activate factor XII in in vitro assays, based on kallikrein release. However, only fucosylated chondroitin sulfate induces hypotension when intravenously injected into rats. In conclusion, the complexity of the regulatory mechanisms involved in the action of highly sulfated polysaccharides in coagulation requires their analysis by a combination of in vitro and in vivo assays. Our results are relevant due to the urgent need for new anticoagulant drugs or alternative sources of heparin.

Combined gelatin-chondroitin sulfate hydrogels for controlled release of cationic antibacterial proteins

NARCIS (Netherlands)

Kuijpers, A. J.; Engbers, G. H. M.; Meyvis, T. K. L.; de Smedt, S. S. C.; Demeester, J.; Krijgsveld, J.; Zaat, S. A. J.; Dankert, J.; Feijen, J.

2000-01-01

Chemically cross-linked gelatin-chondroitin sulfate (ChS) hydrogels were prepared for the controlled release of small cationic proteins. The amount of chondroitin sulfate in the gelatin gels varied between 0 and 20 wt %. The chemical cross-link density, the degree of swelling, and the rheological

Anti-tumor Study of Chondroitin Sulfate-Methotrexate Nanogels

Science.gov (United States)

Wang, Jinyu; Zhao, Weibo; Chen, Haixiao; Qin, An; Zhu, Peizhi

2017-10-01

Self-assembly nanogels (NGs) were formed by bioconjugating methotrexate (MTX) with chondroitin sulfate (CS). MTX-CS NGs can greatly enhance the solubility and improve the delivery efficacy of MTX due to the CD44 binding property of CS. Vivo experiments revealed that MTX-CS NGs showed less toxicity than MTX. MTX-CS NGs can improve the anti-tumor effect while reducing the side effects of MTX. Due to their CD44 binding property, chondroitin sulfate-drug conjugates could be a promising and efficient platform for improving the solubility of sparingly soluble drug molecules as well as targeted delivery to cancer cells and tumor tissues.

Biosynthesis and function of chondroitin sulfate.

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Mikami, Tadahisa; Kitagawa, Hiroshi

2013-10-01

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions. Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo. Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes. Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

Platelet lysate and chondroitin sulfate loaded contact lenses to heal corneal lesions.

Science.gov (United States)

Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Delfino, Alessio; Riva, Federica; Icaro Cornaglia, Antonia; Marrubini, Giorgio; Musitelli, Giorgio; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

2016-07-25

Hemoderivative tear substitutes contain various ephiteliotrophic factors, such as growth factors (GF), involved in ocular surface homeostasis without immunogenic properties. The aim of the present work was the loading of platelet lysate into contact lenses to improve the precorneal permanence of platelet lysate growth factors on the ocular surface to enhance the treatment of corneal lesions. To this purpose, chondroitin sulfate, a sulfated glycosaminoglycan, which is normally present in the extracellular matrix, was associated with platelet lysate. In fact, chondroitin sulfate is capable of electrostatic interaction with positively charged growth factors, in particular, with bFGF, IGF, VEGF, PDGF and TGF-β, resulting in their stabilization and reduced degradation in solution. In the present work, various types of commercially available contact lenses have been loaded with chondroitin sulfate or chondroitin sulfate in association with platelet lysate to achieve a release of growth factors directly onto the corneal surface lesions. One type of contact lenses (PureVision(®)) showed in vitro good proliferation properties towards corneal cells and were able to enhance cut closure in cornea constructs. Copyright © 2016 Elsevier B.V. All rights reserved.

Impaired Autophagy in the Lipid-Storage Disorder Niemann-Pick Type C1 Disease

OpenAIRE

Sarkar, Sovan; Carroll, Bernadette; Buganim, Yosef; Maetzel, Dorothea; Ng, Alex H.M.; Cassady, John P.; Cohen, Malkiel A.; Chakraborty, Souvik; Wang, Haoyi; Spooner, Eric; Ploegh, Hidde; Gsponer, Joerg; Korolchuk, Viktor I.; Jaenisch, Rudolf

2013-01-01

Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal prot...

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library

DEFF Research Database (Denmark)

Sugiura, Nobuo; Clausen, Thomas Mandel; Shioiri, Tatsuasa

2016-01-01

with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental......-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain...... that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation...

Biphasic role of chondroitin sulfate in cardiac differentiation of embryonic stem cells through inhibition of Wnt/β-catenin signaling.

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Robert D Prinz

Full Text Available The glycosaminoglycan chondroitin sulfate is a critical component of proteoglycans on the cell surface and in the extracellular matrix. As such, chondroitin sulfate side chains and the sulfation balance of chondroitin play important roles in the control of signaling pathways, and have a functional importance in human disease. In contrast, very little is known about the roles of chondroitin sulfate molecules and sulfation patterns during mammalian development and cell lineage specification. Here, we report a novel biphasic role of chondroitin sulfate in the specification of the cardiac cell lineage during embryonic stem cell differentiation through modulation of Wnt/beta-catenin signaling. Lineage marker analysis demonstrates that enzymatic elimination of endogenous chondroitin sulfates leads to defects specifically in cardiac differentiation. This is accompanied by a reduction in the number of beating cardiac foci. Mechanistically, we show that endogenous chondroitin sulfate controls cardiac differentiation in a temporal biphasic manner through inhibition of the Wnt/beta-catenin pathway, a known regulatory pathway for the cardiac lineage. Treatment with a specific exogenous chondroitin sulfate, CS-E, could mimic these biphasic effects on cardiac differentiation and Wnt/beta-catenin signaling. These results establish chondroitin sulfate and its sulfation balance as important regulators of cardiac cell lineage decisions through control of the Wnt/beta-catenin pathway. Our work suggests that targeting the chondroitin biosynthesis and sulfation machinery is a novel promising avenue in regenerative strategies after heart injury.

Biphasic role of chondroitin sulfate in cardiac differentiation of embryonic stem cells through inhibition of Wnt/β-catenin signaling.

Science.gov (United States)

Prinz, Robert D; Willis, Catherine M; van Kuppevelt, Toin H; Klüppel, Michael

2014-01-01

The glycosaminoglycan chondroitin sulfate is a critical component of proteoglycans on the cell surface and in the extracellular matrix. As such, chondroitin sulfate side chains and the sulfation balance of chondroitin play important roles in the control of signaling pathways, and have a functional importance in human disease. In contrast, very little is known about the roles of chondroitin sulfate molecules and sulfation patterns during mammalian development and cell lineage specification. Here, we report a novel biphasic role of chondroitin sulfate in the specification of the cardiac cell lineage during embryonic stem cell differentiation through modulation of Wnt/beta-catenin signaling. Lineage marker analysis demonstrates that enzymatic elimination of endogenous chondroitin sulfates leads to defects specifically in cardiac differentiation. This is accompanied by a reduction in the number of beating cardiac foci. Mechanistically, we show that endogenous chondroitin sulfate controls cardiac differentiation in a temporal biphasic manner through inhibition of the Wnt/beta-catenin pathway, a known regulatory pathway for the cardiac lineage. Treatment with a specific exogenous chondroitin sulfate, CS-E, could mimic these biphasic effects on cardiac differentiation and Wnt/beta-catenin signaling. These results establish chondroitin sulfate and its sulfation balance as important regulators of cardiac cell lineage decisions through control of the Wnt/beta-catenin pathway. Our work suggests that targeting the chondroitin biosynthesis and sulfation machinery is a novel promising avenue in regenerative strategies after heart injury.

Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling.

Science.gov (United States)

Ramachandra, Rashmi; Namburi, Ramesh B; Ortega-Martinez, Olga; Shi, Xiaofeng; Zaia, Joseph; Dupont, Sam T; Thorndyke, Michael C; Lindahl, Ulf; Spillmann, Dorothe

2014-02-01

Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates.

Properties of aqueous dispersion of chitosan and chondroitin sulfate complex derived from aquatic organisms

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Novikov V. Yu.

2016-09-01

Full Text Available Investigation of production of chondroitin sulfate, chitosan and polyelectrolyte complexes based on them received from the local marine raw materials is relevant from the point of view of developing a comprehensive waste-free technology for natural raw materials processing. The objects of study are chitosan derived from the shell of the Kamchatka crab Paralithodes camtschaticus and chondroitin sulfate derived from cartilage of salmon Salmon salar. To determine the surface tension of polyelectrolyte complex solutions and dispersions the Wilhelmy method has been used, the effective radius of particle dispersion has been calculated by light scattering, measurements of effective viscosity have been carried out under shear deformation. The conditions of formation, surface and rheological properties of the chitosan and chondroitin sulfate complex extracted from aquatic organisms in the Barents Sea have been studied. Obtaining conditions and molar ratios of these polyelectrolytes in which the aqueous dispersion of the complex remains stable for a long time have been established. It has been found that by addition of chondroitin sulfate solution to chitosan solution in molar ratios of 1 : 3; 1 : 6 the dispersion of the polyelectrolyte complex stable for 2 to 3 days has been formed. The polyelectrolyte complex dispersions behave as non-Newtonian pseudoplastic liquid. When the molar ratio of the mixed solution is 1 : 1 (regardless of the sequence of mixing suspension of the polyelectrolyte complex has been formed, then there is precipitation. Equilibrium surface tension of the aqueous dispersion of the polyelectrolyte complex is higher than that of solutions of chondroitin sulfate and chitosan. The effective radius of particles in the complex dispersion has been determined. The effective radius of the particles in the complex dispersion depends on the molar ratio of chondroitin sulfate : chitosan. A qualitative scheme of formation of polyelectrolyte

Cloning and characterization of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from a marine bacterium.

Science.gov (United States)

Wang, Wenshuang; Han, Wenjun; Cai, Xingya; Zheng, Xiaoyu; Sugahara, Kazuyuki; Li, Fuchuan

2015-03-20

Sulfatases are potentially useful tools for structure-function studies of glycosaminoglycans (GAGs). To date, various GAG exosulfatases have been identified in eukaryotes and prokaryotes. However, endosulfatases that act on GAGs have rarely been reported. Recently, a novel HA and CS lyase (HCLase) was identified for the first time from a marine bacterium (Han, W., Wang, W., Zhao, M., Sugahara, K., and Li, F. (2014) J. Biol. Chem. 289, 27886-27898). In this study, a putative sulfatase gene, closely linked to the hclase gene in the genome, was recombinantly expressed and characterized in detail. The recombinant protein showed a specific N-acetylgalactosamine-4-O-sulfatase activity that removes 4-O-sulfate from both disaccharides and polysaccharides of chondroitin sulfate (CS)/dermatan sulfate (DS), suggesting that this sulfatase represents a novel endosulfatase. The novel endosulfatase exhibited maximal reaction rate in a phosphate buffer (pH 8.0) at 30 °C and effectively removed 17-65% of 4-O-sulfates from various CS and DS and thus significantly inhibited the interactions of CS and DS with a positively supercharged fluorescent protein. Moreover, this endosulfatase significantly promoted the digestion of CS by HCLase, suggesting that it enhances the digestion of CS/DS by the bacterium. Therefore, this endosulfatase is a potential tool for use in CS/DS-related studies and applications. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Histamine and chondroitin sulfate E proteoglycan released by cultured human colonic mucosa: indication for possible presence of E mast cells

International Nuclear Information System (INIS)

Eliakim, R.; Gilead, L.; Ligumsky, M; Okon, E.; Rachmilewitz, D.; Razin, E.

1986-01-01

An association between the release of histamine and chondroitin sulfate E proteoglycan (PG) was demonstrates in human colonic mucosa (HCM). Colonic biopsy samples incorporated [ 35 S]sulfate into PG, which was partially released into the culture medium during the incubation period. Ascending thin-layer chromatography of the released 35 S-labeled PG after its digestion by chondroitin ABC lyase (chondroitinase, EC 4.2.2.4) followed by autoradiography yielded three products that migrated in the position of monosulfated disaccharides of N-acetylgalactosamine 4-sulfate and N-acetylgalactosoamine 6-sulfate and of an oversulfated disaccharide possessing N-acetylgalatosamine 4,6-disulfate. Cultured colonic mucosa released 23.6 +/- 3.7ng of histamine per mg of wet tissue without any special trigger. Comparison by linear regression analysis of the release of histamine and chondroitin [ 35 S]sulfate E PG revealed a correlation coefficient (r) of 0.7. Histological examination of the colonic biopsies revealed the presence of many mast cells in various degrees of degranulation in the mucosa and submucosa. The above correlation, the observation that most of the mast cells showed various degrees of degranulation, and the lack of heparin synthesis as opposed to the synthesis and immunological release of chondroitin sulfate E strongly suggest that the E mast cell exists in the human colon

Amino acid sequence surrounding the chondroitin sulfate attachment site of thrombomodulin regulates chondroitin polymerization.

Science.gov (United States)

Izumikawa, Tomomi; Kitagawa, Hiroshi

2015-05-01

Thrombomodulin (TM) is a cell-surface glycoprotein and a critical mediator of endothelial anticoagulant function. TM exists as both a chondroitin sulfate (CS) proteoglycan (PG) form and a non-PG form lacking a CS chain (α-TM); therefore, TM can be described as a part-time PG. Previously, we reported that α-TM bears an immature, truncated linkage tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl). However, the biosynthetic mechanism to generate part-time PGs remains unclear. In this study, we used several mutants to demonstrate that the amino acid sequence surrounding the CS attachment site influences the efficiency of chondroitin polymerization. In particular, the presence of acidic residues surrounding the CS attachment site was indispensable for the elongation of CS. In addition, mutants defective in CS elongation did not exhibit anti-coagulant activity, as in the case with α-TM. Together, these data support a model for CS chain assembly in which specific core protein determinants are recognized by a key biosynthetic enzyme involved in chondroitin polymerization. Copyright © 2015 Elsevier Inc. All rights reserved.

Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

Science.gov (United States)

Shioiri, Tatsumasa; Tsuchimoto, Jun; Watanabe, Hideto; Sugiura, Nobuo

2016-06-01

Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Inflammatory microRNA-194 and -515 attenuate the biosynthesis of chondroitin sulfate during human intervertebral disc degeneration.

Science.gov (United States)

Hu, Bo; Xu, Chen; Tian, Ye; Shi, Changgui; Zhang, Ying; Deng, Lianfu; Zhou, Hongyu; Cao, Peng; Chen, Huajiang; Yuan, Wen

2017-07-25

Intervertebral disc degeneration (IDD) is characterized by dehydration and loss of extracellular matrixes in the nucleus pulposus region. Chondroitin sulfate has been found to be the water-binding molecule that played a key role in IDD. Although investigators have reported that inflammatory cytokines are involved in the reduction of chondroitin sulfate in IDD, but the underlying mechanism is unrevealed. Since chondroitin sulfate synthesis is controlled by chondroitin sulfate glycosyltransferases CHSY-1/2/3 and CSGALNACT-1/2, their functional role and regulatory mechanism in IDD is not fully studied. Here, we set out to investigate the function and regulatory roles of these factors during IDD development. We found that among these chondroitin sulfate glycosyltransferases, CHSY-1/2/3 are significantly down-regulated in severe IDD samples than mild IDD samples. In vitro experiments revealed that Interleukin-1β and Tumor Necrosis Factor-α stimulation led to significant reduction of CHSY-1/2/3 at protein level than mRNA level in NP cells, indicating a post-transcriptional regulatory mechanisms are involved. By computational prediction and analysis, we found that inflammatory cytokines stimulated microRNA-194 and -515 target CHSY-1/2/3 mRNA and significantly interrupt their translation and downstream chondroitin sulfate deposition. Inhibition of microRNA-194 and -515 however, significantly rescued CHSY-1/2/3 expressions and chondroitin sulfate deposition. These findings together demonstrated a vital role of inflammatory stimulated microRNAs in promoting intervertebral disc degeneration by interrupt chondroitin sulfate synthesis, which may provide new insights into the mechanism and therapeutic approaches in IDD.

Chondroitin sulfate/dermatan sulfate sulfatases from mammals and bacteria.

Science.gov (United States)

Wang, Shumin; Sugahara, Kazuyuki; Li, Fuchuan

2016-12-01

Sulfatases that specifically catalyze the hydrolysis of the sulfate groups on chondroitin sulfate (CS)/dermatan sulfate (DS) poly- and oligosaccharides belong to the formylglycine-dependent family of sulfatases and have been widely found in various mammalian and bacterial organisms. However, only a few types of CS/DS sulfatase have been identified so far. Recently, several novel CS/DS sulfatases have been cloned and characterized. Advanced studies have provided significant insight into the biological function and mechanism of action of CS/DS sulfatases. Moreover, further studies will provide powerful tools for structural and functional studies of CS/DS as well as related applications. This article reviews the recent progress in CS/DS sulfatase research and is expected to initiate further research in this field.

Incorporation of 35S-sulfate and 3H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells

International Nuclear Information System (INIS)

Blair, O.C.; Sartorelli, A.C.

1984-01-01

Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35 S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35 S-sulfate and 3 H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M

Adult Niemann-Pick disease type B with myositis ossificans: a case report

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Russka Shumnalieva

2016-07-01

Full Text Available Niemann-Pick Disease (NPD is a rare autosomal recessive lysosomal lipid storage disorder. The disease is caused by gene mutations that affect the metabolism of sphingolipids. The dysfunctions cause sphingomyelin to accumulate in different organs. NPD includes forms with low and high levels of sphingomyelin. We report a case of a 34 year-old man with a family history of NPD type B who presented with hepatosplenomegaly, neurological deficiency, bone abnormalities, and myositis ossificans. The clinical, biochemical, and imaging data confirmed the combined diagnosis of NPD type B with myositis ossificans.

Rapid Diagnosis of 83 Patients with Niemann Pick Type C Disease and Related Cholesterol Transport Disorders by Cholestantriol Screening

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Janine Reunert

2016-02-01

Full Text Available Niemann Pick type C (NP-C is a rare neurodegenerative disorder caused by an impairment of intracellular lipid transport. Due to the heterogeneous clinical phenotype and the lack of a reliable blood test, diagnosis and therapy are often delayed for years. In the cell, accumulating cholesterol leads to increased formation of oxysterols that can be used as a powerful screening parameter for NP-C. In a large scale study, we evaluated the oxysterol cholestane-3β,5α,6β-triol (c-triol as potential biomarker for a rapid diagnosis of NP-C. Using GC/MS, c-triol has been analyzed in 1902 plasma samples of patients with the suspicion for NP-C. Diagnosis in patients with elevated oxysterols was confirmed by genetic analysis. 71 new NP-C patients (69 NP-C1 and two NP-C2 and 12 Niemann Pick type A/B patients were identified. 24 new mutations in NPC1, one new mutation in NPC2 and three new mutations in the SMPD1 gene were found. Cholestane-3β,5α,6β-triol was elevated in Niemann Pick type C1, type C2, type A/B and in CESD disease. No other study has ever identified so many NP-C patients, proving that c-triol is a rapid and reliable biomarker to detect patients with NP-C disease and related cholesterol transport disorders. It should replace the filipin test as the first-line diagnostic assay.

Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

International Nuclear Information System (INIS)

Stevens, R.L.; Austen, K.F.; Fox, C.C.; Lichtenstein, L.M.

1988-01-01

The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of 35 S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although [ 35 S]heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate [ 35 S]sulfate into separate heparin and chondroitin sulfate proteoglycans in an ∼2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin [ 35 S]sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin [ 35 S]sulfate E proteoglycans and the [ 35 S]heparin proteoglycans were exocytosed from the [ 35 S]sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of 35 S-labeled proteoglycans reside in the secretory granules of these human lung mast cells

Hematopoietic stem cell transplantation in Niemann-Pick disease type B monitored by chitotriosidase activity.

Science.gov (United States)

Quarello, Paola; Spada, Marco; Porta, Francesco; Vassallo, Elena; Timeus, Fabio; Fagioli, Franca

2018-02-01

Here, we report a patient with Niemann-Pick disease type B, with early severe onset of disease and pulmonary involvement, treated with hematopoietic stem cell transplant (HSCT) from a bone marrow matched unrelated donor. We confirm that HSCT is feasible and potentially beneficial for patients with severe phenotype. Noteworthy, we discussed the potential usefulness of the activity of peripheral chitotriosidase for the longitudinal evaluation of HSCT success and effectiveness. © 2017 Wiley Periodicals, Inc.

Chondroitin-6-sulfate-containing proteoglycan: a new component of human skin dermoepidermal junction

DEFF Research Database (Denmark)

Fine, J D; Couchman, J R

1988-01-01

chondroitin sulfate proteoglycan is present in adult, neonatal, and/or fetal skin, and if present, its ultrastructural localization. Indirect immunofluorescence was performed on human adult, neonatal, and fetal skin. To detect the antigen, specimens were pretreated with chondroitinase ABC; absence of enzyme...... treatment served as negative control. Chondroitin sulfate proteoglycan was detectable in linear homogeneous array along the dermoepidermal junction and within vascular (and when present, adnexal) basement membranes in both adult and neonatal skin. In fetal skin, basement membrane staining was noted as early...... as 54 gestational days. Indirect immunoelectron microscopy and NaCl-split skin studies were performed to ultrastructurally localize the antigen; immune deposits were detectable within the lamina densa in chondroitinase-treated skin. These findings demonstrate that chondroitin sulfate proteoglycan...

Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates.

Science.gov (United States)

Numakura, Mario; Kusakabe, Noriko; Ishige, Kazuya; Ohtake-Niimi, Shiori; Habuchi, Hiroko; Habuchi, Osami

2010-07-01

Chondroitin sulfate (CS) containing GlcA-GalNAc(4,6-SO(4)) (E unit) and CS containing GlcA(2SO(4))-GalNAc(6SO(4)) (D unit) have been implicated in various physiological functions. However, it has been poorly understood how the structure and contents of disulfated disaccharide units in CS contribute to these functions. We prepared CS libraries containing E unit or D unit in various proportions by in vitro enzymatic reactions using recombinant GalNAc 4-sulfate 6-O-sulfotransferase and uronosyl 2-O-sulfotransferase, and examined their inhibitory activity toward thrombin. The in vitro sulfated CSs containing disulfated disaccharide units showed concentration-dependent direct inhibition of thrombin when the proportion of E unit or D unit in the CSs was above 15-17%. The CSs containing both E unit and D unit exhibited higher inhibitory activity toward thrombin than the CSs containing either E unit or D unit alone, if the proportion of the total disulfated disaccharide units of these CSs was comparable. The thrombin-catalyzed degradation of fibrinogen, a physiological substrate for thrombin, was also inhibited by the CS containing both E unit and D unit. These observations indicate that the enzymatically prepared CS libraries containing various amounts of disulfated disaccharide units appear to be useful for elucidating the physiological function of disulfated disaccharide units in CS.

Biphasic Role of Chondroitin Sulfate in Cardiac Differentiation of Embryonic Stem Cells through Inhibition of Wnt/beta-Catenin Signaling

NARCIS (Netherlands)

Prinz, R.D.; Willis, C.M.; Kuppevelt, T.H. van; Kluppel, M.

2014-01-01

The glycosaminoglycan chondroitin sulfate is a critical component of proteoglycans on the cell surface and in the extracellular matrix. As such, chondroitin sulfate side chains and the sulfation balance of chondroitin play important roles in the control of signaling pathways, and have a functional

Peripheral neuropathy in type A Niemann-Pick disease. A morphological study.

Science.gov (United States)

Landrieu, P; Saïd, G

1984-01-01

A black boy had a severe neuropathic form of Niemann-Pick disease (NPD) with a pronounced sphingomyelinase deficiency in the fibroblasts. Nerve conduction velocities were diminished, and a nerve biopsy was performed. Isolated fibers showed segmental demyelination and numerous dense bodies in the Schwann cells (SC). Electron microscopy revealed two categories of inclusions: the first was made up of lysosomal inclusions usually described in NPD. The second comprised myelin inclusions--sometimes still connected to the original myelin sheath--indicating severe myelinopathy. Both myelin debris and NPD inclusions were found in axoplasms and probably came from SC cytoplasm through axolemma lesions. NPD is a unique example of myelinopathy due to sphingomyelinase deficiency.

Extraction and determination of chondroitin sulfate from fish processing byproducts

Science.gov (United States)

Chondroitin sulfate (CS) refers to a group of sulfated glycosaminoglycan containing a chain of alternating N-acetylgalactosamine and glucuronic acid sugars. It is a major component of the extracellular matrix of cartilage and attached to proteins. CS is usually an over the counter dietary supplement...

Chondroitin Sulfate (CS) Lyases: Structure, Function and Application in Therapeutics.

Science.gov (United States)

Rani, Aruna; Patel, Seema; Goyal, Arun

2018-01-01

Glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) are the chief natural polysaccharides which reside in biological tissues mainly in extracellular matrix. These CS along with adhesion molecules and growth factors are involved in central nervous system (CNS) development, cell progression and pathogenesis. The chondroitin lyases are the enzyme that degrade and alter the CS chains and hence modify various signalling pathways involving CS chains. These CS lyases are substrate specific, can precisely manipulate the CS polysaccharides and have various biotechnological, medical and therapeutic applications. These enzymes can be used to produce the unsaturated oligosaccharides, which have immune-modulatory, anti-inflammatory and neuroprotective properties. This review focuses on the major breakthrough of the chondroitin sulfate degrading enzymes, their structures and functioning mechanism. This also provides comprehensive information regarding production, purification, characterization of CS lyases and their major applications, both established as well as emerging ones such as neural development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Intestinal mucosal mast cells from rats infected with Nippostrongylus brasiliensis contain protease-resistant chondroitin sulfate di-B proteoglycans

International Nuclear Information System (INIS)

Stevens, R.L.; Lee, T.D.G.; Seldin, D.C.; Austen, K.F.; Befus, A.D.; Bienenstock, J.

1986-01-01

Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [ 35 S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35 S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The isolated proteoglycans were of approx. 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched populations of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leumekia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans are all highly sulfated, protease-resistant proteoglycans

Adult onset Niemann-Pick type C disease: A clinical, neuroimaging and molecular genetic study.

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Battisti, Carla; Tarugi, Patrizla; Dotti, Maria Teresa; De Stefano, Nicola; Vattimo, Angelo; Chierichetti, Francesea; Calandra, Sebastiano; Federico, Antonio

2003-11-01

We report on a patient with adult-onset Niemann-Pick type C (NPC) disease, carrying the mutations P1007 and I1061T in the NPC1 gene, presenting with marked psychiatric changes followed by dystonia and cognitive impairment. Filipin staining, single photon emission computed tomography perfusional, positron emission tomography metabolic, conventional magnetic resonance imaging, and magnetic resonance spectroscopy findings suggested a pathophysiological correlation with phenotype expression. This case expands the clinical and genetic spectrum of the rare adult-onset NPC disease phenotype.

Liver and Skin Histopathology in Adults with Acid Sphingomyelinase Deficiency (Niemann-Pick Disease Type B)

OpenAIRE

Thurberg, Beth L.; Wasserstein, Melissa P.; Schiano, Thomas; O’Brien, Fanny; Richards, Susan; Cox, Gerald F.; McGovern, Margaret M.

2012-01-01

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder characterized by the pathologic accumulation of sphingomyelin in multiple cells types, and occurs most prominently within the liver, spleen and lungs, leading to significant clinical disease. Seventeen ASMD patients underwent a liver biopsy during baseline screening for a Phase 1 trial of recombinant human acid sphingomyelinase (rhASM) in adults with Niemann-Pick disease type B. Eleven of the 17 were enrolled in the trial...

Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans

International Nuclear Information System (INIS)

Calvo, J.C.; Rodbard, D.; Katki, A.; Chernick, S.; Yanagishita, M.

1991-01-01

The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with [35S]sulfate and [3H] glucosamine for 24 h and then extracted and analyzed. There was a 1.68 ± 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of ∼ 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of ∼ 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 ± 0.2-fold in media and 3.2 ± 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation

Extracellular matrix of cultured glial cells: Selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

International Nuclear Information System (INIS)

Gallo, V.; Bertolotto, A.

1990-01-01

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space

Ultrastructural immunocytochemical localization of chondroitin sulfate proteoglycan in Bruch's membrane of the rat

DEFF Research Database (Denmark)

Lin, W L; Essner, E; McCarthy, K J

1992-01-01

Two monoclonal antibodies (Mab 4D5 and 2D6) raised against the core protein of a basement membrane chondroitin sulfate proteoglycan from Reichert's membrane of the rat, were used for ultrastructural immunoperoxidase localization of this protein in Bruch's membrane of the rat. Immunoreactivity...

Impact of miglustat on evolution of atypical presentation of late-infantile-onset Niemann?Pick disease type C with early cognitive impairment, behavioral dysfunction, epilepsy, ophthalmoplegia, and cerebellar involvement: a case report

OpenAIRE

Cuisset, Jean-Marie; Sukno, S.; Trauffler, A.; Latour, P.; Dobbelaere, D.; Michaud, L.; Vall?e, L.

2016-01-01

Background Niemann?Pick disease type C is a rare inherited neurodegenerative disease involving impaired intracellular lipid trafficking and accumulation of glycolipids in various tissues, including the brain. Miglustat, a reversible inhibitor of glucosylceramide synthase, has been shown to be effective in the treatment of progressive neurological manifestations in pediatric and adult patients with Niemann?Pick disease type C, and has been used in that indication in Europe since 2010. Case pre...

Do chondroitin sulfates with different structures have different activities on chondrocytes and macrophages?

Science.gov (United States)

da Cunha, André L; Aguiar, Jair A K; Correa da Silva, Flavio S; Michelacci, Yara M

2017-10-01

The aim of the present study was to investigate the activities of natural chondroitin sulfates (CS) with different structures on cultured chondrocytes and macrophages. CS were isolated from cartilages of bovine trachea (BT), porcine trachea (PT), chicken sternum (Ch) and skate (Sk). The preparations were 90-98% pure, with ∼1% proteins, nucleic acids and keratan sulfate contaminants. Structural analysis of these CS and of commercial chondroitin 4- and 6-sulfate (C4S, C6S) have shown that most of their disaccharides are monosulfated, with varying proportions of 4- and 6-sulfation, and 2-7% non-sulfated disaccharides. Sk-CS and C6S contained detectable amounts of disulfated disaccharides. All the CS were polydisperse, with modal molecular weights of 26-135kDa. These CS had anti-inflammatory activities on both chondrocytes and macrophages, but with different efficiencies. On horse and human chondrocytes, they reduced the IL-1β-induced liberation of NO and PGE 2 , and on RAW 264.7 immortalized macrophage-like cell line, C4S, C6S, Ch and Sk-CS decreased the LPS-induced liberation of TNF-α, but did not affect IL-6. In contrast, on bone marrow derived macrophages, C4S, C6S, BT and PT-CS reduced the LPS-induced liberation of TNF-α, IL-6, IL-1β and NO, indicating that the RAW response to CS was different from that of primary macrophages. Copyright © 2017 Elsevier B.V. All rights reserved.

Medical Gains of Chondroitin Sulfate Upon Fucosylation.

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Pomin, Vitor H

2015-01-01

Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) composed of alternating N-acetyl galactosamine and glucuronic acid units within disaccharide building blocks. CS is a key functional component in proteoglycans of cartilaginous tissues. Owing to its numerous biological roles, CS is widely explored in the pharmaceutical market as nutraceutical ingredient commonly utilized against arthritis, osteoarthrosis, and sometimes osteoporosis. Tissues like shark cartilage and bovine trachea are common sources of CS. Nonetheless, a new CS type has been introduced and investigated in the last few decades in what regards its medical potentials. It is named fucosylated chondroitin sulfate (FucCS). This less common CS type is isolated exclusively from the body wall of sea cucumbers. The presence of fucosyl branching units in the holothurian FucCS gives to this unique GAG, therapeutic properties in various pathophysiological systems which are inexistent in the common CS explored in the market. Examples of these systems are coagulation, thrombosis, hemodialysis, atherosclerosis, cellular growth, angiogenesis, fibrosis, tumor growth, inflammation, viral and protozoan infections, hyperglycemia, diabetes-related pathological events and tissue damage. This report aims at describing the medical benefits gained upon fucosylation of CS. Clinical prospects of these medical benefits are also discussed herein.

Sphingomyelin lipidosis (Niemann-Pick disease) in a juvenile raccoon (Procyon lotor).

Science.gov (United States)

Vapniarsky, N; Wenger, D A; Scheenstra, D; Mete, A

2013-01-01

A wild caught juvenile male raccoon with neurological disease was humanely destroyed due to poor prognosis. Necropsy examination revealed hepatomegaly, splenomegaly and multicentric lymphadenomegaly with diffuse hepatic pallor and pulmonary consolidation with pinpoint pale subpleural foci. Microscopically, there was marked pale cytoplasmic swelling of the central and peripheral neurons as well as the glial cells in the brain, accompanied by multiorgan infiltration by abundant foamy macrophages. Ultrastructural investigation revealed accumulation of concentrically arranged lamellar material within lysosomes of the affected neurons, macrophages and endothelial cells. Biochemical enzymatic analysis detected sphingomyelinase deficiency and lysosomal storage disease consistent with sphingomyelin lipidosis (Niemann-Pick disease [NPD]) was diagnosed. This is the first report of NPD in a raccoon. Copyright © 2013 Elsevier Ltd. All rights reserved.

Induced Pluripotent Stem Cells for Disease Modeling and Evaluation of Therapeutics for Niemann-Pick Disease Type A.

Science.gov (United States)

Long, Yan; Xu, Miao; Li, Rong; Dai, Sheng; Beers, Jeanette; Chen, Guokai; Soheilian, Ferri; Baxa, Ulrich; Wang, Mengqiao; Marugan, Juan J; Muro, Silvia; Li, Zhiyuan; Brady, Roscoe; Zheng, Wei

2016-12-01

: Niemann-Pick disease type A (NPA) is a lysosomal storage disease caused by mutations in the SMPD1 gene that encodes acid sphingomyelinase (ASM). Deficiency in ASM function results in lysosomal accumulation of sphingomyelin and neurodegeneration. Currently, there is no effective treatment for NPA. To accelerate drug discovery for treatment of NPA, we generated induced pluripotent stem cells from two patient dermal fibroblast lines and differentiated them into neural stem cells. The NPA neural stem cells exhibit a disease phenotype of lysosomal sphingomyelin accumulation and enlarged lysosomes. By using this disease model, we also evaluated three compounds that reportedly reduced lysosomal lipid accumulation in Niemann-Pick disease type C as well as enzyme replacement therapy with ASM. We found that α-tocopherol, δ-tocopherol, hydroxypropyl-β-cyclodextrin, and ASM reduced sphingomyelin accumulation and enlarged lysosomes in NPA neural stem cells. Therefore, the NPA neural stem cells possess the characteristic NPA disease phenotype that can be ameliorated by tocopherols, cyclodextrin, and ASM. Our results demonstrate the efficacies of cyclodextrin and tocopherols in the NPA cell-based model. Our data also indicate that the NPA neural stem cells can be used as a new cell-based disease model for further study of disease pathophysiology and for high-throughput screening to identify new lead compounds for drug development. Currently, there is no effective treatment for Niemann-Pick disease type A (NPA). To accelerate drug discovery for treatment of NPA, NPA-induced pluripotent stem cells were generated from patient dermal fibroblasts and differentiated into neural stem cells. By using the differentiated NPA neuronal cells as a cell-based disease model system, α-tocopherol, δ-tocopherol, and hydroxypropyl-β-cyclodextrin significantly reduced sphingomyelin accumulation in these NPA neuronal cells. Therefore, this cell-based NPA model can be used for further study of

Purification, structural characterization and anticoagulant properties of fucosylated chondroitin sulfate isolated from Holothuria mexicana.

Science.gov (United States)

Mou, Jiaojiao; Wang, Cong; Li, Wenjing; Yang, Jie

2017-05-01

A novel fucosylated chondroitin sulfate (HmG) was isolated from sea cucumber Holothuria mexicana, the structure of which was characterized by monosaccharide composition, disaccharide composition, IR, 1 H and 13 C NMR spectrum, additionally with two dimensional NMR spectrum of degraded HmG (DHmG). The backbone of HmG was identified as chondroitin 6-O sulfate, while the major O-4 sulfated fucose branches linked to O-3 position of glucuronic acid in almost every disaccharide unit. The anticoagulant activities of HmG and DHmG were assessed and compared with heparin and low molecular weight heparin. The results indicated that HmG and DHmG both could significantly prolong the activated partial thrombo-plastin time, and the properties were well related to its molecular weight. DHmG showed similar anticoagulant properties to low molecular weight heparin with less bleeding risks, making it a safer anticoagulant drug. Copyright © 2017 Elsevier B.V. All rights reserved.

Sequencing of chondroitin sulfate oligosaccharides using a novel exolyase from a marine bacterium that degrades hyaluronan and chondroitin sulfate/dermatan sulfate.

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Wang, Wenshuang; Cai, Xiaojuan; Han, Naihan; Han, Wenjun; Sugahara, Kazuyuki; Li, Fuchuan

2017-11-09

Glycosaminoglycans (GAGs) are a family of chemically heterogeneous polysaccharides that play important roles in physiological and pathological processes. Owing to the structural complexity of GAGs, their sophisticated chemical structures and biological functions have not been extensively studied. Lyases that cleave GAGs are important tools for structural analysis. Although various GAG lyases have been identified, exolytic lyases with unique enzymatic property are urgently needed for GAG sequencing. In the present study, a putative exolytic GAG lyase from a marine bacterium was recombinantly expressed and characterized in detail. Since it showed exolytic lyase activity toward hyaluronan (HA), chondroitin sulfate (CS), and dermatan sulfate (DS), it was designated as HCDLase. This novel exolyase exhibited the highest activity in Tris-HCl buffer (pH 7.0) at 30°C. Especially, it showed a specific activity that released 2-aminobenzamide (2-AB)-labeled disaccharides from the reducing end of 2-AB-labeled CS oligosaccharides, which suggest that HCDLase is not only a novel exolytic lyase that can split disaccharide residues from the reducing termini of sugar chains but also a useful tool for the sequencing of CS chains. Notably, HCDLase could not digest 2-AB-labeled oligosaccharides from HA, DS, or unsulfated chondroitin, which indicated that sulfates and bond types affect the catalytic activity of HCDLase. Finally, this enzyme combined with CSase ABC was successfully applied for the sequencing of several CS hexa- and octasaccharides with complex structures. The identification of HCDLase provides a useful tool for CS-related research and applications. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Preparation and characterization of hydroxyapatite/chondroitin sulfate composites by biomimetic synthesis

International Nuclear Information System (INIS)

Xiao Xiufeng; He Dan; Liu Fang; Liu Rongfang

2008-01-01

Based on the principles of biomineralization, flakelike hydroxyapatite/chondroitin sulfate composites were synthesized through biomimetic method using Ca(NO 3 ) 2 .4H 2 O and (NH 4 ) 3 PO 4 .3H 2 O as reagents and chondroitin sulfate as template. The crystalline phase, microstructure, chemical composition, morphology and thermal behavior of the composites obtained in the experiment were characterized by means of X-ray diffraction (XRD), Fourier transform infrared spectroscope (FTIR), transmission electron microscope (TEM), Thermogravimetry-Differential thermal analyzer (TG-DTA) and Elemental analyzer, respectively. The interaction between the functional groups of ChS and HA was investigated by electrical conductivity and UV-vis spectrum. The results demonstrate that the as-prepared powders with small amount of carbonate have the component similar to human bone. It can be concluded that the nucleation and growth of HA crystals occurred through the chemical interactions between the HA crystals and preorganized functional groups of the ChS template. Furthermore, the concentration of ChS significantly affects the morphology of the composites. Short fiberlike crystals could be obtained at a low concentration of ChS, but flakelike crystals could be synthesized using a high concentration (≥0.5 wt%) of ChS as template

Preparation and characterization of hydroxyapatite/chondroitin sulfate composites by biomimetic synthesis

Energy Technology Data Exchange (ETDEWEB)

Xiao Xiufeng; He Dan; Liu Fang [College of Chemistry and Materials Science, Fujian Normal University, Fujian, Fuzhou 350007 (China); Liu Rongfang [College of Chemistry and Materials Science, Fujian Normal University, Fujian, Fuzhou 350007 (China)], E-mail: rfliu@vip.sina.com

2008-12-20

Based on the principles of biomineralization, flakelike hydroxyapatite/chondroitin sulfate composites were synthesized through biomimetic method using Ca(NO{sub 3}){sub 2}.4H{sub 2}O and (NH{sub 4}){sub 3}PO{sub 4}.3H{sub 2}O as reagents and chondroitin sulfate as template. The crystalline phase, microstructure, chemical composition, morphology and thermal behavior of the composites obtained in the experiment were characterized by means of X-ray diffraction (XRD), Fourier transform infrared spectroscope (FTIR), transmission electron microscope (TEM), Thermogravimetry-Differential thermal analyzer (TG-DTA) and Elemental analyzer, respectively. The interaction between the functional groups of ChS and HA was investigated by electrical conductivity and UV-vis spectrum. The results demonstrate that the as-prepared powders with small amount of carbonate have the component similar to human bone. It can be concluded that the nucleation and growth of HA crystals occurred through the chemical interactions between the HA crystals and preorganized functional groups of the ChS template. Furthermore, the concentration of ChS significantly affects the morphology of the composites. Short fiberlike crystals could be obtained at a low concentration of ChS, but flakelike crystals could be synthesized using a high concentration ({>=}0.5 wt%) of ChS as template.

Chondroitin / dermatan sulfate modification enzymes in zebrafish development.

Directory of Open Access Journals (Sweden)

Judith Habicher

Full Text Available Chondroitin/dermatan sulfate (CS/DS proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS. Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.

Orbitrap mass spectrometry characterization of hybrid chondroitin/dermatan sulfate hexasaccharide domains expressed in brain.

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Robu, Adrian C; Popescu, Laurentiu; Munteanu, Cristian V A; Seidler, Daniela G; Zamfir, Alina D

2015-09-15

In the central nervous system, chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) modulate neurotrophic effects and glial cell maturation during brain development. Previous reports revealed that GAG composition could be responsible for CS/DS activities in brain. In this work, for the structural characterization of DS- and CS-rich domains in hybrid GAG chains extracted from neural tissue, we have developed an advanced approach based on high-resolution mass spectrometry (MS) using nanoelectrospray ionization Orbitrap in the negative ion mode. Our high-resolution MS and multistage MS approach was developed and applied to hexasaccharides obtained from 4- and 14-week-old mouse brains by GAG digestion with chondroitin B and in parallel with AC I lyase. The expression of DS- and CS-rich domains in the two tissues was assessed comparatively. The analyses indicated an age-related structural variability of the CS/DS motifs. The older brain was found to contain more structures and a higher sulfation of DS-rich regions, whereas the younger brain was found to be characterized by a higher sulfation of CS-rich regions. By multistage MS using collision-induced dissociation, we also demonstrated the incidence in mouse brain of an atypical [4,5-Δ-GlcAGalNAc(IdoAGalNAc)2], presenting a bisulfated CS disaccharide formed by 3-O-sulfate-4,5-Δ-GlcA and 6-O-sulfate-GalNAc moieties. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library.

Science.gov (United States)

Sugiura, Nobuo; Clausen, Thomas Mandel; Shioiri, Tatsumasa; Gustavsson, Tobias; Watanabe, Hideto; Salanti, Ali

2016-12-01

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-D-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.

Chondroitin 6-O-sulfate ameliorates experimental autoimmune encephalomyelitis.

Science.gov (United States)

Miyamoto, Katsuichi; Tanaka, Noriko; Moriguchi, Kota; Ueno, Rino; Kadomatsu, Kenji; Kitagawa, Hiroshi; Kusunoki, Susumu

2014-05-01

Chondroitin sulfate proteoglycans (CSPGs) are the main component of the extracellular matrix in the central nervous system (CNS) and influence neuroplasticity. Although CSPG is considered an inhibitory factor for nerve repair in spinal cord injury, it is unclear whether CSPG influences the pathogenetic mechanisms of neuroimmunological diseases. We induced experimental autoimmune encephalomyelitis (EAE) in chondroitin 6-O-sulfate transferase 1-deficient (C6st1(-/-)) mice. C6ST1 is the enzyme that transfers sulfate residues to position 6 of N-acetylgalactosamine in the sugar chain of CSPG. The phenotypes of EAE in C6st1(-/-) mice were more severe than those in wild-type (WT) mice were. In adoptive-transfer EAE, in which antigen-reactive T cells from WT mice were transferred to C6st1(-/-) and WT mice, phenotypes were significantly more severe in C6st1(-/-) than in WT mice. The recall response of antigen-reactive T cells was not significantly different among the groups. Furthermore, the number of pathogenic T cells within the CNS was also not considerably different. When EAE was induced in C6ST1 transgenic mice with C6ST1 overexpression, the mice showed considerably milder symptoms compared with those in WT mice. In conclusion, the presence of sulfate at position 6 of N-acetylgalactosamine of CSPG may influence the effecter phase of EAE to prevent the progression of pathogenesis. Thus, modification of the carbohydrate residue of CSPG may be a novel therapeutic strategy for neuroimmunological diseases such as multiple sclerosis.

Structural characterization of fucosylated chondroitin sulfates from sea cucumbers Apostichopus japonicus and Actinopyga mauritiana.

Science.gov (United States)

Ustyuzhanina, Nadezhda E; Bilan, Maria I; Dmitrenok, Andrey S; Tsvetkova, Eugenia A; Shashkov, Alexander S; Stonik, Valentin A; Nifantiev, Nikolay E; Usov, Anatolii I

2016-11-20

Two samples of fucosylated chondroitin sulfate (FCS), AJ and AM, were isolated from holothurian species Apostichopus japonicus and Actinopyga mauritiana, respectively. Purification of FCS was performed by ion exchange chromatography followed by gel filtration. Structure of the biopolymers was elucidated using chemical and NMR spectroscopic methods. Both polysaccharides were shown to contain a typical chondroitin core built up of repeating disaccharide units →3)-β-d-GalNAc-(1→4)-β-d-GlcA-(1→ and decorated by sulfate groups and α-l-Fuc branches. Two polysaccharides were different in pattern of sulfation of GalNAc and fucosyl branches connected to O-3 of GlcA. The ratio of GalNAc4S6S:GalNAc4S for AJ was about 2:1, whereas for AM this value was approximately 1:1. AJ contained Fucp2S4S and Fucp3S4S residues linked to O-3 of GlcA in a ratio of 3:1, while for AM this ratio was 1:4. Small portions of Fucp4S units attached to O-3 of GlcA were also found in both polysaccharides. Moreover, in a structure of AM the presence of Fucp3S residues linked to O-6 of GalNAc were determined using the data of NMR spectra. Copyright © 2016 Elsevier Ltd. All rights reserved.

PEG-lipid micelles enable cholesterol efflux in Niemann-Pick Type C1 disease-based lysosomal storage disorder

OpenAIRE

Brown, Anna; Patel, Siddharth; Ward, Carl; Lorenz, Anna; Ortiz, Mauren; DuRoss, Allison; Wieghardt, Fabian; Esch, Amanda; Otten, Elsje G.; Heiser, Laura M.; Korolchuk, Viktor I.; Sun, Conroy; Sarkar, Sovan; Sahay, Gaurav

2016-01-01

2-Hydroxy-propyl-?-cyclodextrin (HP?CD), a cholesterol scavenger, is currently undergoing Phase 2b/3 clinical trial for treatment of Niemann Pick Type C-1 (NPC1), a fatal neurodegenerative disorder that stems from abnormal cholesterol accumulation in the endo/lysosomes. Unfortunately, the extremely high doses of HP?CD required to prevent progressive neurodegeneration exacerbates ototoxicity, pulmonary toxicity and autophagy-based cellular defects. We present unexpected evidence that a poly (e...

Chondroitin Sulfate Proteoglycan 4 and Its Potential As an Antibody Immunotherapy Target across Different Tumor Types

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Kristina M. Ilieva

2018-01-01

Full Text Available Overexpression of the chondroitin sulfate proteoglycan 4 (CSPG4 has been associated with the pathology of multiple types of such as melanoma, breast cancer, squamous cell carcinoma, mesothelioma, neuroblastoma, adult and pediatric sarcomas, and some hematological cancers. CSPG4 has been reported to exhibit a role in the growth and survival as well as in the spreading and metastasis of tumor cells. CSPG4 is overexpressed in several malignant diseases, while it is thought to have restricted and low expression in normal tissues. Thus, CSPG4 has become the target of numerous anticancer treatment approaches, including monoclonal antibody-based therapies. This study reviews key potential anti-CSPG4 antibody and immune-based therapies and examines their direct antiproliferative/metastatic and immune activating mechanisms of action.

[Sensitive Determination of Chondroitin Sulfate by Fluorescence Recovery of an Anionic Aluminum Phthalocyanine-Cationic Surfactant Ion-Association Complex Used as a Fluorescent Probe Emitting at Red Region].

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Chen, Lin; Huang, Ping; Yang, Hui-qing; Deng, Ya-bin; Guo, Meng-lin; Li, Dong-hui

2015-08-01

Determination of chondroitin sulfate in the biomedical field has an important value. The conventional methods for the assay of chondroitin sulfate are still unsatisfactory in sensitivity, selectivity or simplicity. This work aimed at developing a novel method for sensitive and selective determination of chondroitin sulfate by fluorimetry. We found that some kinds of cationic surfactants have the ability to quench the fluorescence of tetrasulfonated aluminum phthalocyanine (AlS4Pc), a strongly fluorescent compound which emits at red region, with high efficiency. But, the fluorescence of the above-mentioned fluorescence quenching system recovered significantly when chondroitin sulfate (CS) exits. Tetradecyl dimethyl benzyl ammonium chloride(TDBAC) which was screened from all of the candidates of cationic surfactants was chosen as the quencher because it shows the most efficient quenching effect. It was found that the fluorescence of AlS4Pc was extremely quenched by TDBAC because of the formation of association complex between AlS4Pc and TDBAC. Fluorescence of the association complex recovered dramatically after the addition of chondroitin sulfate (CS) due to the ability of chondroitin sulfate to shift the association equilibrium of the association, leading to the release of AlS4Pc, thus resulting in an increase in the fluorescence of the reaction system. Based on this phenomenon, a novel method with simplicity, accuracy and sensitivity was developed for quantitative determination of CS. Factors including the reaction time, influencing factors and the effect of coexisting substances were investigated and discussed. Under optimum conditions the linear range of the calibration curve was 0.20~10.0 μg · mL(-1). The detection limit for CS was 0.070 μg · mL(-1). The method has been applied to the analysis of practical samples with satisfied results. This work expands the applications of AlS4Pc in biomedical area.

Is chondroitin sulfate responsible for the biological effects attributed to the GC protein-derived Macrophage Activating Factor (GcMAF)?

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Ruggiero, Marco; Reinwald, Heinz; Pacini, Stefania

2016-09-01

We hypothesize that a plasma glycosaminoglycan, chondroitin sulfate, may be responsible for the biological and clinical effects attributed to the Gc protein-derived Macrophage Activating Factor (GcMAF), a protein that is extracted from human blood. Thus, Gc protein binds chondroitin sulfate on the cell surface and such an interaction may occur also in blood, colostrum and milk. This interpretation would solve the inconsistencies encountered in explaining the effects of GcMAF in vitro and in vivo. According to our model, the Gc protein or the GcMAF bind to chondroitin sulfate both on the cell surface and in bodily fluids, and the resulting multimolecular complexes, under the form of oligomers trigger a transmembrane signal or, alternatively, are internalized and convey the signal directly to the nucleus thus eliciting the diverse biological effects observed for both GcMAF and chondroitin sulfate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Xyloside-primed Chondroitin Sulfate/Dermatan Sulfate from Breast Carcinoma Cells with a Defined Disaccharide Composition Has Cytotoxic Effects in Vitro.

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Persson, Andrea; Tykesson, Emil; Westergren-Thorsson, Gunilla; Malmström, Anders; Ellervik, Ulf; Mani, Katrin

2016-07-08

We previously reported that the xyloside 2-(6-hydroxynaphthyl) β-d-xylopyranoside (XylNapOH), in contrast to 2-naphthyl β-d-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH- and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH- and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells. The cytotoxic effect appeared to be mediated by induction of apoptosis and was inhibited in a concentration-dependent manner by the XylNap-primed heparan sulfate GAGs. In contrast, neither the chondroitin sulfate/dermatan sulfate nor the heparan sulfate derived from CCD-1095Sk cells primed on XylNapOH or XylNap had any effect on the growth of HCC70 cells or CCD-105Sk cells. These observations were related to the disaccharide composition of the XylNapOH- and XylNap-primed GAGs, which differed between the two cell lines but was similar when the GAGs were derived from the same cell line. To our knowledge this is the first report on cytotoxic effects mediated by chondroitin sulfate/dermatan sulfate. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Spectrum of SMPD1 mutations in Asian-Indian patients with acid sphingomyelinase (ASM)-deficient Niemann-Pick disease.

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Ranganath, Prajnya; Matta, Divya; Bhavani, Gandham SriLakshmi; Wangnekar, Savita; Jain, Jamal Mohammed Nurul; Verma, Ishwar C; Kabra, Madhulika; Puri, Ratna Dua; Danda, Sumita; Gupta, Neerja; Girisha, Katta M; Sankar, Vaikom H; Patil, Siddaramappa J; Ramadevi, Akella Radha; Bhat, Meenakshi; Gowrishankar, Kalpana; Mandal, Kausik; Aggarwal, Shagun; Tamhankar, Parag Mohan; Tilak, Preetha; Phadke, Shubha R; Dalal, Ashwin

2016-10-01

Acid sphingomyelinase (ASM)-deficient Niemann-Pick disease is an autosomal recessive lysosomal storage disorder caused by biallelic mutations in the SMPD1 gene. To date, around 185 mutations have been reported in patients with ASM-deficient NPD world-wide, but the mutation spectrum of this disease in India has not yet been reported. The aim of this study was to ascertain the mutation profile in Indian patients with ASM-deficient NPD. We sequenced SMPD1 in 60 unrelated families affected with ASM-deficient NPD. A total of 45 distinct pathogenic sequence variants were found, of which 14 were known and 31 were novel. The variants included 30 missense, 4 nonsense, and 9 frameshift (7 single base deletions and 2 single base insertions) mutations, 1 indel, and 1 intronic duplication. The pathogenicity of the novel mutations was inferred with the help of the mutation prediction software MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA. The effects of the identified sequence variants on the protein structure were studied using the structure modeled with the help of the SWISS-MODEL workspace program. The p. (Arg542*) (c.1624C>T) mutation was the most commonly identified mutation, found in 22% (26 out of 120) of the alleles tested, but haplotype analysis for this mutation did not identify a founder effect for the Indian population. To the best of our knowledge, this is the largest study on mutation analysis of patients with ASM-deficient Niemann-Pick disease reported in literature and also the first study on the SMPD1 gene mutation spectrum in India. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Visualization of liver on bone scintigraphy during treatment with iron chondroitin sulfate colloid

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Tanaka, Shigeko; Sawa, Hisashi; Fukuda, Teruo

1983-09-01

In order to see its probable cause in the incidental liver delineation on the bone scan with sup(99m)Tc-MDP, patients and experimental rabbits were intravenously injected with Blutal (iron chondroitin sulfate) at various times following the intravenous injection of sup(99m)Tc-MDP. The liver images were obtained during the early periods. An injection of Blutal-sup(99m)Tc pertechnetate mixture did not result in any appreciable hepatic delineation. Stannous chloride in the MDP kit could have enticed formation of radiocolloids in the presence of iron chondroitin sulfate. Diagnostic problems can be avoided by carefully planning the bone scintigraphy in relation to the Blutal administration. (author).

Comparison of Glucosamine-Chondroitin Sulfate with and without Methylsulfonylmethane in Grade I-II Knee Osteoarthritis: A Double Blind Randomized Controlled Trial.

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Lubis, Andri M T; Siagian, Carles; Wonggokusuma, Erick; Marsetyo, Aldo F; Setyohadi, Bambang

2017-04-01

Glucosamine, chondroitinsulfate are frequently used to prevent further joint degeneration in osteoarthritis (OA). Methylsulfonylmethane (MSM) is a supplement containing organic sulphur and also reported to slow anatomical joint progressivity in the knee OA. The MSM is often combined with glucosamine and chondroitin sulfate. However, there are controversies whether glucosamine-chondroitin sulfate or their combination with methylsulfonylmethane could effectively reduce pain in OA. This study is aimed to compare clinical outcome of glucosamine-chondroitin sulfate (GC), glucosamine-chondroitin sulfate-methylsulfonylmethane (GCM), and placeboin patients with knee osteoarthritis (OA) Kellgren-Lawrence grade I-II. a double blind, randomized controlled clinical trial was conducted on 147 patients with knee OA Kellgren-Lawrence grade I-II. Patients were allocated by permuted block randomization into three groups: GC (n=49), GCM (n=50), or placebo (n=48) groups. GC group received 1500 mg of glucosamine + 1200 mg of chondroitin sulfate + 500 mg of saccharumlactis; GCM group received 1500 mg of glucosamine + 1200 mg of chondroitin sulfate + 500 mg of MSM; while placebo group received three matching capsules of saccharumlactis. The drugs were administered once daily for 3 consecutive months VAS and WOMAC scores were measured before treatment, then at 4th, 8th and 12th week after treatment. on statistical analysis it was found that at the 12th week, there are significant difference between three treatment groups on the WOMAC score (p=0.03) and on the VAS score (p=0.004). When analyzed between weeks, GCM treatment group was found statistically significant on WOMAC score (p=0.01) and VAS score (p<0.001). Comparing the score difference between weeks, WOMAC score analysis showed significant difference between GC, GCM, and placebo in week 4 (p=0.049) and week 12 (p=0.01). In addition, VAS score also showed significant difference between groups in week 8 (p=0.006) and week 12 (p<0

Synthesis of Fucosylated Chondroitin Sulfate Glycoclusters: A Robust Route to New Anticoagulant Agents.

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Zhang, Xiao; Yao, Wang; Xu, Xiaojiang; Sun, Huifang; Zhao, Jinhua; Meng, Xiangbao; Wu, Mingyi; Li, Zhongjun

2018-02-01

Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan with excellent anticoagulant activity. Studies show that FuCS and its depolymerized fragments exhibit a different anticoagulant mechanism from that of heparin derivatives, with decreased risks of adverse effects and bleeding. However, further exploitation has been hindered by the scarcity of structurally defined oligosaccharides. Herein, facile method is reported for the synthesis of the repeating trisaccharide unit of FuCS based on the degradation of chondroitin sulfate polymers. A series of simplified FuCS glycomimetics that have highly tunable structures, controllable branches, and defined sulfation motifs were generated by copper-catalyzed alkyne-azide cycloaddition. Remarkable improvement in activated partial thromboplastin time (APTT) assay activities was observed as the branches increased, but no significant influences were observed for prothrombin time (PT) and thrombin time (TT) assay activities. Further FXase inhibition tests suggested that glycoclusters 33 b-40 b selectively inhibited intrinsic anticoagulant activities, but had little effect on the extrinsic and common coagulation pathways. Notably, glycoclusters with the 2,4-di-O-sulfated fucosyl residue displayed the most potency, which was in consistent with that of natural polysaccharides. These FuCS clusters demonstrated potency to mimic linear glycosaminoglycans and offer a new framework for the development of novel anticoagulant agents. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

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Shuji Mizumoto

2013-01-01

Full Text Available Chondroitin sulfate (CS containing E-disaccharide units, glucuronic acid-N-acetylgalactosamine(4, 6-O-disulfate, at surfaces of tumor cells plays a key role in tumor metastasis. However, the molecular mechanism of the metastasis involving the CS chain-containing E-units is not fully understood. In this study, to clarify the role of E-units in the metastasis and to search for potential molecular targets for anticancer drugs, the isolation and characterization of Lewis lung carcinoma (LLC cells stably downregulated by the knockdown for the gene encoding N-acetylgalactosamine 4-O-sulfate 6-O-sulfotransferase (GalNAc4S-6ST, which is responsible for the formation of E-units in CS chains, were performed. Knockdown of GalNAc4S-6ST in LLC cells resulted in a reduction in the proportion of E-units, in adhesiveness to extracellular matrix adhesion molecules and in proliferation in vitro. Furthermore, the stable downregulation of GalNAc4S-6ST expression in LLC cells markedly inhibited the colonization of the lungs by inoculated LLC cells and invasive capacity of LLC cells. These results provide clear evidence that CS chain-containing E-units and/or GalNAc4S-6ST play a crucial role in pulmonary metastasis at least through the increased adhesion and the invasive capacity of LLC cells and also provides insights into future drug targets for anticancer treatment.

Structure and biological activity of a fucosylated chondroitin sulfate from the sea cucumber Cucumaria japonica.

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Ustyuzhanina, Nadezhda E; Bilan, Maria I; Dmitrenok, Andrey S; Shashkov, Alexander S; Kusaykin, Mikhail I; Stonik, Valentin A; Nifantiev, Nikolay E; Usov, Anatolii I

2016-05-01

A fucosylated chondroitin sulfate (FCS) was isolated from the body wall of Pacific sea cucumber Cucumaria japonicaby extraction in the presence of papain followed by Cetavlon precipitation and anion-exchange chromatography. FCS was shown to contain D-GalNAc, D-GlcA, L-Fuc and sulfate in molar proportions of about 1:1:1:4.5. Structure of FCS was elucidated using NMR spectroscopy and methylation analysis of the native polysaccharide and products of its desulfation and carboxyl reduction. The polysaccharide was shown to contain a typical chondroitin core → 3)-β-D-GalNAc-(1 → 4)-β-D-GlcA-(1 →. Sulfate groups in this core occupy O-4 and the majority of O-6 of GalNAc. Fucosyl branches are represented by 3,4- and 2,4-disulfated units in a ratio of 4:1 and are linked to O-3 of GlcA. In addition, ∼ 33% of GlcA are 3-O-sulfated, and hence, the presence of short fucooligosaccharide chains side by side with monofucosyl branches cannot be excluded. FCS was shown to inhibit platelets aggregation in vitro mediated by collagen and ristocetin, but not adenosine diphosphate, and demonstrated significant anticoagulant activity, which is connected with its ability to enhance inhibition of thrombin and factor Xa by antithrombin III, as well as to influence von Willebrand factor activity. The latest property significantly distinguished FCS from low-molecular-weight heparin. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Distinct Niemann-Pick Disease Type C Clinical, Cytological, and Biochemical Phenotype in an Adult Patient With 1 Mutated, Overexpressed Allele

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Julia Jecel MD

2015-11-01

Full Text Available Niemann-Pick disease type C (NP-C is a rare autosomal-recessive neurovisceral lysosomal storage disease. We report on a juvenile onset, now 25-year-old female patient with typical neurologic symptoms, including vertical gaze palsy, of NP-C. The diagnosis was supported by a positive filipin test (“variant biochemical phenotype” of cholesterol accumulation in cultured fibroblasts, high numbers of “Niemann-Pick cells” in the bone marrow, and 1 positive out of 3 NP-C biomarkers tested, but NP-C was not definitely confirmed genetically. She showed only 1 known NPC1 variant (3 bp deletion in exon 18; p.N916del; this allele, however, being distinctly overexpressed at the messenger RNA level as compared to the wild-type allele, as a not as yet clarified (copathogenic? phenomenon. The patient’s mother, also carrying the p.N916del allele but without overexpression, has a chronic inflammatory disease of the central nervous system classified as multiple sclerosis. However, her severe clinical phenotype includes some signs also consistent with NP-C. The laboratory diagnosis of NP-C can be challenging in detecting novel disease constellations.

Effectiveness of intra-articular injections of sodium hyaluronate-chondroitin sulfate in knee osteoarthritis: a multicenter prospective study.

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Rivera, Fabrizio; Bertignone, Luca; Grandi, Giancarlo; Camisassa, Roberto; Comaschi, Guido; Trentini, Diego; Zanone, Marco; Teppex, Giuseppe; Vasario, Gabriele; Fortina, Giorgio

2016-03-01

Intra-articular injection of hyaluronic acid is a well-established therapy for the treatment of knee osteoarthritis. The aim of the study was to assess the effectiveness and safety of the use of Arthrum HCS(®) (40 mg hyaluronic acid and 40 mg chondroitin sulfate in 2 mL). This was an open, multicenter, prospective study. Men or women over 40 years of age with documented knee osteoarthritis and WOMAC subscore A (severity of pain) ≥25 were enrolled. They received three weekly intra-articular injections of sodium hyaluronate 2 % and chondroitin sulfate 2 % in combination. WOMAC subscore A was assessed at 1, 3 and 6 months after the last injection. One hundred and twelve patients were included (women, 66 %). The mean (SD) WOMAC subscore A decreased from 52.1 (15.2) at inclusion to 20.5 (19.7) at month 6 (P chondroitin sulfate in reducing pain (77 %), improving mobility (78 %) and reducing the consumption of analgesics (74 %). Only one adverse effect was reported by one patient (knee tumefaction). These results suggest that intra-articular injections of Arthrum HCS(®) (sodium hyaluronate plus chondroitin sulfate) in patients with knee osteoarthritis are efficient and safe. These results should be confirmed in a randomized controlled study. IV.

Differentiating chondroitin sulfate glycosaminoglycans using collision-induced dissociation; uronic acid cross-ring diagnostic fragments in a single stage of tandem mass spectrometry.

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Kailemia, Muchena J; Patel, Anish B; Johnson, Dane T; Li, Lingyun; Linhardt, Robert J; Amster, I Jonathan

2015-01-01

The stereochemistry of the hexuronic acid residues of the structure of glycosaminoglycans (GAGs) is a key feature that affects their interactions with proteins and other biological functions. Electron based tandem mass spectrometry methods, in particular electron detachment dissociation (EDD), have been able to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) residues in some heparan sulfate tetrasaccharides by producing epimer-specific fragments. Similarly, the relative abundance of glycosidic fragment ions produced by collision-induced dissociation (CID) or EDD has been shown to correlate with the type of hexuronic acid present in chondroitin sulfate GAGs. The present work examines the effect of charge state and degree of sodium cationization on the CID fragmentation products that can be used to distinguish GlcA and IdoA containing chondroitin sulfate A and dermatan sulfate chains. The cross-ring fragments (2,4)A(n) and (0,2)X(n) formed within the hexuronic acid residues are highly preferential for chains containing GlcA, distinguishing it from IdoA. The diagnostic capability of the fragments requires the selection of a molecular ion and fragment ions with specific ionization characteristics, namely charge state and number of ionizable protons. The ions with the appropriate characteristics display diagnostic properties for all the chondroitin sulfate and dermatan sulfate chains (degree of polymerization of 4-10) studied.

Vestibular function in patients with Niemann-Pick type C disease.

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Bremova, Tatiana; Krafczyk, Siegbert; Bardins, Stanislavs; Reinke, Jörg; Strupp, Michael

2016-11-01

We investigated whether vestibular dysfunction may cause or contribute to postural imbalance and falls in patients with Niemann-Pick type C disease (NP-C). Eight patients with NP-C disease and 20 healthy controls were examined using the video-based head impulse test (vHIT) and caloric irrigation to investigate horizontal canal function as well as ocular- and cervical vestibular evoked myogenic potentials (o- and cVEMP), and binocular subjective visual vertical estimation (SVV) for otolith function, and static posturography. There were no significant differences in vestibulo-ocular gain, caloric excitability, o-/cVEMP measures or SVV between the two groups. Posturographic total sway path (tSP) and root mean square (RMS) were significantly higher in NP-C than in controls in 3 out of 4 conditions. The Romberg quotient (RQ) to assess the amount of visual stabilization was significantly lower in the NP-C than in the HC group. In contrast to other inherited metabolic disorders, such as Morbus Gaucher type 3, we did not find any evidence for an impairment of canal or otolith function in patients with NP-C as their cause of postural imbalance. Since RQ was low in NP-C patients, indicating proper sensory input, the observed increased postural sway is most likely due to a cerebellar dysfunction in NP-C, which may therefore, explain postural imbalance.

Conformational studies on five octasaccharides isolated from chondroitin sulfate using NMR spectroscopy and molecular modeling

NARCIS (Netherlands)

Blanchard, V.; Chevalier, F.; Imberty, A.; Leeflang, B.R.; Sugahara, K.; Kamerling, J.P.

2007-01-01

Chondroitin sulfate proteoglycans (CS-PG) are involved in the regulation of the central nervous system in vertebrates due to their presence on cell surfaces and in the extracellular matrix of tissues. The CS moieties are built up from repeating -4)GlcA(β 1-3)GalNAc(β 1- disaccharide units, partly

sup(113m)indium-iron chondroitin sulfate colloid for quantitative assessment of the marrow RE function

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Okuyama, S; Ito, Y; Takahashi, K; Sato, T; Matsuzawa, T [Tohoku Univ., Sendai (Japan). Research Inst. for Tuberculosis, Leprosy and Cancer

1975-07-01

sup(113m)In-iron chondroitin sulfate colloid shows a large accumulation in the bone marrow and is suitable for bone marrow imaging. Quantitative assessment of the marrow reticuloendotherial function was performed using this compound. When an appropriate amount of iron carrier was added for adjustment, the rate of accumulation of hyperfunction in the marrow reticuloendotherial system (RES) induced by acute loss of blood increased. Marrow RES hypofunction was efficiently exhibited regardless of the presence or absence of iron carrier. Deposition of sup(113m)In-iron chondroitin sulfate in the spleen increased remarkably in the presence of carrier In. sup(113m)In-iron chondroitin sulfate colloid appears to be suitable for the measurement of the conditions of marrow RES functions. If short half-life nuclide radio-colloids of the present type are clinically applied, it is possible not only to elaborately observe the bone marrow by scintigraphy but also to gradually decrease the absorbed dose of irradiation.

sup(113m)indium-iron chondroitin sulfate colloid for quantitative assessment of the marrow RE function

International Nuclear Information System (INIS)

Okuyama, Shinichi; Ito, Yasuhiko; Takahashi, Kunibumi; Sato, Tachio; Matsuzawa, Taiju

1975-01-01

sup(113m)In-iron chondroitin sulfate colloid shows a large accumulation in the bone marrow and is suitable for bone marrow imaging. Quantitative assessment of the marrow reticuloendotherial function was performed using this compound. When an appropriate amount of iron carrier was added for adjustment, the rate of accumulation of hyperfunction in the marrow reticuloendotherial system (RES) induced by acute loss of blood increased. Marrow RES hypofunction was efficiently exhibited regardless of the presence or absence of iron carrier. Deposition of sup(113m)In-iron chondroitin sulfate in the spleen increased remarkably in the presence of carrier In. sup(113m)In-iron chondroitin sulfate colloid appears to be suitable for the measurement of the conditions of marrow RES functions. If short half-life nuclide radio-colloids of the present type are clinically applied, it is possible not only to elaborately observe the bone marrow by scintigraphy but also to gradually decrease the absorbed dose of irradiation. (Mukohata, S.)

An efficency of use phonophoresis with an ointment on the basis of chondroitin sulfate and dimetil sulfoxide at the treatment of patients with arthritis of knee joints

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Виктор Александрович Вишневский

2015-06-01

sulfate and dimethyl sulfoxide in patients with osteoarthritis allows increase an efficiency of treatment by 20 % in comparison with standard scheme of physiotherapy in the control group.3.      Positive dynamics on the background of treatment was confirmed by the 2 international tests and laboratory examination of the level of oxyproline in daily urine and also by the more fast disappearance or decrease of pain syndrome and by increase of joints function in patients of the main group.4.      The use of phonophoresis with an ointment on the basis of chondroitin sulfates and dimethyl sulfoxide on injured knee joint is prescribed not only for inpatient treatment but also for an outpatient one at aftercare of residual effects especially in persons of an elderly age as a more available method that allows increase the quality of life in patients

Oncofetal Chondroitin Sulfate Glycosaminoglycans are Key Players in Integrin Signaling and Tumor Cell Motility

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Clausen, Thomas Mandel; Pereira, Marina Ayres; Al Nakouzi, Nader; Oo, Htoo Zarni; Agerbæk, Mette Ø; Lee, Sherry; Ørum-Madsen, Maj Sofie; Christensen, Anders Riis; El-Naggar, Amal; Grandgenett, Paul M.; Grem, Jean L.; Hollingsworth, Michael A.; Holst, Peter J.; Theander, Thor; Sorensen, Poul H.; Daugaard, Mads; Salanti, Ali

2016-01-01

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum. We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion and anchorage-independent growth of tumor cells in vitro. Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns, revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin β1 (ITGB1) and integrin α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core CS synthesis enzymes Beta-1,3-Glucuronyltransferase 1 (B3GAT1) and Chondroitin Sulfate N-Acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and pre-incubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. Implications The cancer specific expression of oncofetal chondroitin sulfate aids in metastatic phenotypes and is a candidate target for therapy. PMID:27655130

Purification and characterization of a 3'-phosphoadenylylsulfate:chondroitin 6-sulfotransferase from arterial tissue.

Science.gov (United States)

Hollmann, J; Niemann, R; Buddecke, E

1986-01-01

A 3'-phosphoadenylylsulfate:chondroitin sulfotransferase (EC 2.8.2.5) was purified to homogeneity (about 760-fold) from the cytosolic fraction of calf arterial tissue by Con A-Sepharose, ion exchange and affinity chromatography. The enzyme has a molecular mass of 38000 Da, optimal activity at pH 6.0 (100%) and 7.25 (75%), requires divalent cations for maximal activity (Mn2+ greater than Mg2+, Ca2+) and exhibits specificity towards desulfated chondroitin sulfate and oligosaccharides derived therefrom. The enzyme transfers sulfate groups from [35S]phosphoadenylylsulfate exclusively to C-6 OH groups of N-acetylgalactosamine units of the acceptor substrates. Maximal sulfate transfer occurs at 2mM chondroitin disaccharide units (100%), the transfer rates decreasing with decreasing chain length in the order deca (55%), octa (17%) and hexasaccharides (4%). Lineweaver-Burk plots revealed equal maximal velocities for chondroitin, deca-, octa- and hexasaccharide, but decreasing Km values. Chondroitin 4-sulfate has 21% of the acceptor potency exhibited by chondroitin, whereas dermatan sulfate, heparan sulfate and hyaluronate and the chondroitin tetrasaccharide showed no acceptor properties. Analysis of the reaction products formed by prolonged enzymatic sulfation of a reduced chondroitin hexasaccharide [GlcA-GalNAc]2-GlcA-GalNAc-ol revealed that the preterminal N-acetylgalactosamine from the non-reducing end and the internal N-acetylgalactosamine but not the N-acetylgalactosaminitol were sulfated and that no hexasaccharide disulfate was formed by the action of chondroitin 6-sulfotransferase. Chondroitin 6-sulfotransferase is considered to possess a binding region capable of accommodating a nonsulfated oligosaccharide sequence of at least six sugars and is believed to act in the course of chondroitin sulfate synthesis in cooperation with, but shortly after, the enzymes involved in the chain elongation reaction.

Probable Diagnosis of a Patient with Niemann-Pick Disease Type C: Managing Pitfalls of Exome Sequencing.

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Zeiger, William A; Jamal, Nasheed I; Scheuner, Maren T; Pittman, Patricia; Raymond, Kimiyo M; Morra, Massimo; Mishra, Shri K

2018-02-17

Here, we present a case of a 31-year-old man with progressive cognitive decline, ataxia, and dystonia. Extensive laboratory, radiographic, and targeted genetic studies over the course of several years failed to yield a diagnosis. Initial whole exome sequencing through a commercial laboratory identified several variants of uncertain significance; however, follow-up clinical examination and testing ruled each of these out. Eventually, repeat whole exome sequencing identified a known pathogenic intronic variant in the NPC1 gene (NM_000271.4, c.1554-1009G>A) and an additional heterozygous exonic variant of uncertain significance in the NPC1 gene (NM_000271.4, c.2524T>C). Follow-up biochemical testing was consistent with a diagnosis of probable Niemann-Pick disease Type C (NP-C). This case illustrates the potential of whole exome sequencing for diagnosing rare complex neurologic diseases. It also identifies several potential common pitfalls that must be navigated by clinicians when interpreting commercial whole exome sequencing results.

Structural analysis of isomeric chondroitin sulfate oligosaccharides using regioselective 6-O-desulfation method and tandem mass spectrometry.

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Chen, Shu-Ting; Her, Guor-Rong

2014-09-16

A strategy based on a regioselective 6-O-desulfation reaction and negative ion electrospray ionization tandem mass spectrometry (ESI-MS(n)) was developed for the structural delineation of isomeric chondroitin sulfate oligosaccharides. Product ions resulting from the glycosidic cleavage provided information about the number of sulfate groups in each sugar residue. After the regioselective 6-O-desulfation reaction, the number of sulfate groups on each residue was obtained using a tandem mass spectrometry analysis of the reaction product. The sulfation pattern could be obtained based on the product ions of analytes before and after the desulfation reaction. The strategy was demonstrated using a series of tetrasaccharides prepared from shark cartilage chondroitin sulfate D. Among the 12 identified tetrasaccharides, six structures had not been reported before. Copyright © 2014 Elsevier B.V. All rights reserved.

The effect of desulfation of chondroitin sulfate on interactions with positively charged growth factors and upregulation of cartilaginous markers in encapsulated MSCs.

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Lim, Jeremy J; Temenoff, Johnna S

2013-07-01

Sulfated glycosaminoglycans (GAGs) are known to interact electrostatically with positively charged growth factors to modulate signaling. Therefore, regulating the degree of sulfation of GAGs may be a promising approach to tailor biomaterial carriers for controlled growth factor delivery and release. For this study, chondroitin sulfate (CS) was first desulfated to form chondroitin, and resulting crosslinked CS and chondroitin hydrogels were examined in vitro for release of positively charged model protein (histone) and for their effect on cartilaginous differentiation of encapsulated human mesenchymal stem cells (MSCs). Desulfation significantly increased the release of histone from chondroitin hydrogels (30.6 ± 2.3 μg released over 8 days, compared to natively sulfated CS with 20.2 ± 0.8 μg), suggesting that sulfation alone plays a significant role in modulating protein interactions with GAG hydrogels. MSCs in chondroitin hydrogels significantly upregulated gene expression of collagen II and aggrecan by day 21 in chondrogenic medium (115 ± 100 and 23.1 ± 7.9 fold upregulation of collagen II and aggrecan, respectively), compared to CS hydrogels and PEG-based swelling controls, indicating that desulfation may actually enhance the response of MSCs to soluble chondrogenic cues, such as TGF-β1. Thus, desulfated chondroitin materials present a promising biomaterial tool to further investigate electrostatic GAG/growth factor interactions, especially for repair of cartilaginous tissues. Copyright © 2013 Elsevier Ltd. All rights reserved.

Safety assessment of non-animal chondroitin sulfate sodium: Subchronic study in rats, genotoxicity tests and human bioavailability.

Science.gov (United States)

Miraglia, Niccolò; Bianchi, Davide; Trentin, Antonella; Volpi, Nicola; Soni, Madhu G

2016-07-01

Chondroitin sulfate, an amino sugar polymer made of glucuronic acid and N-acetyl-galactosamine, is used in dietary supplements to promote joint health. Commonly used chondroitin sulfate is of animal origin and can pose potential safety problems including bovine spongiform encephalopathy (BSE). The objective of the present study was to investigate potential adverse effects, if any, of microbial derived chondroitin sulfate sodium (CSS) in subchronic toxicity, genotoxicity and bioavailability studies. In the toxicity study, Sprague Dawley rats (10/sex/group) were gavaged with CSS at dose levels of 0, 250, 500 and 1000 mg/kg body weight (bw)/day for 90-days. No mortality or significant changes in clinical signs, body weights, body weight gain or feed consumption were noted. Similarly, no toxicologically relevant treatment-related changes in hematological, clinical chemistry, urinalysis and organ weights were noted. Macroscopic and microscopic examinations did not reveal treatment-related abnormalities. In vitro mutagenic and clastogenic potentials as evaluated by Ames assay, chromosomal aberration test and micronucleus assay did not reveal genotoxicity of CSS. In pharmacokinetic study in human, CSS showed higher absorption as compared to chondroitin sulfate of animal origin. The results of subchronic toxicity study supports the no-observed-adverse-effect level (NOAEL) for CSS as 1000 mg/kg bw/day, the highest dose tested. Copyright © 2016 Elsevier Ltd. All rights reserved.

The chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from marine bacterium Vibrio sp FC509 is a dimeric species: Biophysical characterization of an endosulfatase.

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Neira, José L; Medina-Carmona, Encarnación; Hernández-Cifre, José G; Montoliu-Gaya, Laia; Cámara-Artigás, Ana; Seffouh, Ilham; Gonnet, Florence; Daniel, Régis; Villegas, Sandra; de la Torre, José García; Pey, Angel L; Li, Fuchuan

2016-12-01

Sulfatases catalyze hydrolysis of sulfate groups. They have a key role in regulating the sulfation states that determine the function of several scaffold molecules. Currently, there are no studies of the conformational stability of endosulfatases. In this work, we describe the structural features and conformational stability of a 4-O-endosulfatase (EndoV) from a marine bacterium, which removes specifically the 4-O-sulfate from chondroitin sulfate/dermatan sulfate. For that purpose, we have used several biophysical techniques, namely, fluorescence, circular dichroism (CD), FTIR spectroscopy, analytical ultracentrifugation (AUC), differential scanning calorimetry (DSC), mass spectrometry (MS), dynamic light scattering (DLS) and size exclusion chromatography (SEC). The protein was a dimer with an elongated shape. EndoV acquired a native-like structure in a narrow pH range (7.0-9.0); it is within this range where the protein shows the maximum of enzymatic activity. The dimerization did not involve the presence of disulphide-bridges as suggested by AUC, SEC and DLS experiments in the presence of β-mercaptoethanol (β-ME). EndoV secondary structure is formed by a mixture of α and β-sheet topology, as judged by deconvolution of CD and FTIR spectra. Thermal and chemical denaturations showed irreversibility and the former indicates that protein did not unfold completely during heating. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Effects of a synthetic bioactive peptide on neurite growth and nerve growth factor release in chondroitin sulfate hydrogels

OpenAIRE

Conovaloff, Aaron W.; Beier, Brooke L.; Irazoqui, Pedro P.; Panitch, Alyssa

2011-01-01

Previous work has revealed robust dorsal root ganglia neurite growth in hydrogels of chondroitin sulfate. In the current work, it was determined whether addition of a synthetic bioactive peptide could augment neurite growth in these matrices via enhanced binding and sequestering of growth factors. Fluorescence recovery after photobleaching studies revealed that addition of peptide slowed nerve growth factor diffusivity in chondroitin sulfate gels, but not in control gels of hyaluronic acid. F...

Divergent Synthesis of Chondroitin Sulfate Disaccharides and Identification of Sulfate Motifs that Inhibit Triple Negative Breast Cancer

Science.gov (United States)

Wei Poh, Zhong; Heng Gan, Chin; Lee, Eric J.; Guo, Suxian; Yip, George W.; Lam, Yulin

2015-09-01

Glycosaminoglycans (GAGs) regulate many important physiological processes. A pertinent issue to address is whether GAGs encode important functional information via introduction of position specific sulfate groups in the GAG structure. However, procurement of pure, homogenous GAG motifs to probe the “sulfation code” is a challenging task due to isolation difficulty and structural complexity. To this end, we devised a versatile synthetic strategy to obtain all the 16 theoretically possible sulfation patterns in the chondroitin sulfate (CS) repeating unit; these include rare but potentially important sulfated motifs which have not been isolated earlier. Biological evaluation indicated that CS sulfation patterns had differing effects for different breast cancer cell types, and the greatest inhibitory effect was observed for the most aggressive, triple negative breast cancer cell line MDA-MB-231.

Purification and sequence characterization of chondroitin sulfate and dermatan sulfate from fishes.

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Lin, Na; Mo, Xiaoli; Yang, Yang; Zhang, Hong

2017-04-01

Chondroitin sulfate (CS) and dermatan sulfate (DS) were extracted and purified from skins or bones of salmon (Salmo salar), snakehead (Channa argus), monkfish (Lophius litulon) and skipjack tuna (Katsuwonus pelamis). Size, structural sequences and sulfate groups of oligosaccharides in the purified CS and DS could be characterized and identified using high performance liquid chromatography (HPLC) combined with Orbitrap mass spectrometry. CS and DS chain structure varies depending on origin, but motif structure appears consistent. Structures of CS and DS oligosaccharides with different size and sulfate groups were compared between fishes and other animals, and results showed that some minor differences of special structures could be identified by hydrophilic interaction chromatography-liquid chromatography-fourier transform-mass/mass spectrometry (HILIC-LC-FT-MS/MS). For example, data showed that salmon and skipjack CS had a higher percentage content of high-level sulfated oligosaccharides than that porcine CS. In addition, structural information of different origins of CS and DS was analyzed by principal component analysis (PCA) and results showed that CS and DS samples could be differentiated according to their molecular conformation and oligosaccharide fragments information. Understanding CS and DS structure derived from different origins may lead to the production of CS or DS with unique disaccharides or oligosaccharides sequence composition and biological functions.

On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis

DEFF Research Database (Denmark)

Holmborn, Katarina; Habicher, Judith; Kasza, Zsolt

2012-01-01

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation...... levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies...

Niemann-Pick disease type C

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Vanier Marie T

2010-06-01

Full Text Available Abstract Niemann-Pick C disease (NP-C is a neurovisceral atypical lysosomal lipid storage disorder with an estimated minimal incidence of 1/120 000 live births. The broad clinical spectrum ranges from a neonatal rapidly fatal disorder to an adult-onset chronic neurodegenerative disease. The neurological involvement defines the disease severity in most patients but is typically preceded by systemic signs (cholestatic jaundice in the neonatal period or isolated spleno- or hepatosplenomegaly in infancy or childhood. The first neurological symptoms vary with age of onset: delay in developmental motor milestones (early infantile period, gait problems, falls, clumsiness, cataplexy, school problems (late infantile and juvenile period, and ataxia not unfrequently following initial psychiatric disturbances (adult form. The most characteristic sign is vertical supranuclear gaze palsy. The neurological disorder consists mainly of cerebellar ataxia, dysarthria, dysphagia, and progressive dementia. Cataplexy, seizures and dystonia are other common features. NP-C is transmitted in an autosomal recessive manner and is caused by mutations of either the NPC1 (95% of families or the NPC2 genes. The exact functions of the NPC1 and NPC2 proteins are still unclear. NP-C is currently described as a cellular cholesterol trafficking defect but in the brain, the prominently stored lipids are gangliosides. Clinical examination should include comprehensive neurological and ophthalmological evaluations. The primary laboratory diagnosis requires living skin fibroblasts to demonstrate accumulation of unesterified cholesterol in perinuclear vesicles (lysosomes after staining with filipin. Pronounced abnormalities are observed in about 80% of the cases, mild to moderate alterations in the remainder ("variant" biochemical phenotype. Genotyping of patients is useful to confirm the diagnosis in the latter patients and essential for future prenatal diagnosis. The differential

Dynamic expression of a native chondroitin sulfate epitope reveals microheterogeneity of extracellular matrix organization in the embryonic chick heart.

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Capehart, A A; Mjaatvedt, C H; Hoffman, S; Krug, E L

1999-02-01

TC2 is a novel monoclonal antibody produced by in vitro immunization of splenocytes with a peanut agglutinin-positive fraction from extracts of prechondrogenic micromass cultures of chick limb mesenchyme. ELISA results demonstrated TC2 reactivity with a native epitope on a glycosaminoglycan (GAG) enriched in chondroitin-4-sulfate and with multiple intact proteoglycans, but not with other GAGs tested. TC2 immunohistochemical reactivity was abolished by pretreatment of sections with chondroitinase AC or preadsorption with chondroitin-4-sulfate GAG. Strong TC2 localization occurred throughout the developing heart at stage 9. As looping ensued, a graded reactivity was observed from lowest in the atrium to highest in the conotruncus that correlated well with versican localization. The superior atrioventricular cushion stained preferentially with TC2 as compared to the inferior cushion at stages 16-18. At these later stages TC2 patterns did not agree completely with anti-versican reactivity. By stage 23 there was a marked reduction in TC2 localization in the heart, however, strong reactivity remained at certain sites, including the conotruncus and in subcompartments of both atrioventricular cushions. A heterogeneous distribution of other native chondroitin sulfate glycosaminoglycan epitopes recognized by monoclonal antibodies d1C4 and CS-56 was observed as well. The distribution of the TC2 epitope usually did not overlap with d1C4 or CS-56 localization at the stages examined. Overall, the spatiotemporal characteristics of TC2 reactivity in the developing chick heart appear to correlate with subdomains of the endocardial cushions as well as with trabecular and atrial septal formation.

Interactions of oversulfated chondroitin sulfate (OSCS) from different sources with unfractionated heparin.

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Gray, Angel; Litinas, Evangelos; Jeske, Walter; Fareed, Jawed; Hoppensteadt, Debra

2012-01-01

In 2008, oversulfated chondroitin sulfate (OSCS) was identified as the main contaminant in recalled heparin. Oversulfated chondroitin sulfate can be prepared from bovine (B), porcine (P), shark (Sh), or skate (S) origin and may produce changes in the antithrombotic, bleeding, and hemodynamic profile of heparins. This study examines the interactions of various OSCSs on heparin in animal models of thrombosis and bleeding, as well as on the anticoagulant and antiprotease effects in in vitro assays. Mixtures of 70% unfractionated heparin (UFH) with 30% OSCS from different sources were tested. In the in vitro activated partial thromboplastin time (aPTT) assay, all contaminant mixtures showed a decrease in clotting times. In addition, a significant increase in bleeding time compared to the control (UFH/saline) was observed. In the thrombosis model, no significant differences were observed. The OSCSs significantly increased anti-Xa activity in ex vivo blood samples. These results indicate that various sources of OSCS affect the hemostatic properties of heparin.

A characteristic chondroitin sulfate trisaccharide unit with a sulfated fucose branch exhibits neurite outgrowth-promoting activity: Novel biological roles of fucosylated chondroitin sulfates isolated from the sea cucumber Apostichopus japonicus.

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Shida, Miharu; Mikami, Tadahisa; Tamura, Jun-Ichi; Kitagawa, Hiroshi

2017-06-03

Chondroitin sulfate (CS) is a class of sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide unit composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). CS chains are found throughout the pericellular and extracellular spaces and contribute to the formation of functional microenvironments for numerous biological events. However, their structure-function relations remain to be fully characterized. Here, a fucosylated CS (FCS) was isolated from the body wall of the sea cucumber Apostichopus japonicus. Its promotional effects on neurite outgrowth were assessed by using isolated polysaccharides and the chemically synthesized FCS trisaccharide β-D-GalNAc(4,6-O-disulfate) (1-4)[α-l-fucose (2,4-O-disulfate) (1-3)]-β-D-GlcA. FCS polysaccharides contained the E-type disaccharide unit GlcA-GalNAc(4,6-O-disulfate) as a CS major backbone structure and carried distinct sulfated fucose branches. Despite their relatively lower abundance of E unit, FCS polysaccharides exhibited neurite outgrowth-promoting activity comparable to squid cartilage-derived CS-E polysaccharides, which are characterized by their predominant E units, suggesting potential roles of the fucose branch in neurite outgrowth. Indeed, the chemically synthesized FCS trisaccharide was as effective as CS-E tetrasaccharide in stimulating neurite elongation in vitro. In conclusion, FCS trisaccharide units with 2,4-O-disulfated fucose branches may provide new insights into understanding the structure-function relations of CS chains. Copyright © 2017 Elsevier Inc. All rights reserved.

Exploring the structure of fucosylated chondroitin sulfate through bottom-up nuclear magnetic resonance and electrospray ionization-high-resolution mass spectrometry approaches.

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Santos, Gustavo Rc; Porto, Ana Co; Soares, Paulo Ag; Vilanova, Eduardo; Mourão, Paulo As

2017-07-01

Fucosylated chondroitin sulfate (FCS) from sea cucumbers is composed of a chondroitin sulfate (CS) central core and branches of sulfated fucose. The structure of this complex glycosaminoglycan is usually investigated via nuclear magnetic resonance (NMR) analyses of the intact molecule, ergo through a top-down approach, which often yield spectra with intricate sets of signals. Here we employed a bottom-up approach to analyze the FCSs from the sea cucumbers Isostichopus badionotus and Ludwigothurea grisea from their basic constituents, viz. CS cores and sulfated fucose branches, obtained via systematic fragmentation through mild acid hydrolysis. Oligosaccharides derived from the central CS core were analyzed via NMR spectroscopy and the disaccharides produced using chondroitin sulfate lyase via SAX-HPLC. The CS cores from the two species were similar, showing only slight differences in the proportions of 4- or 6-monosulfated and 4,6-disulfated β-d-GalNAc. Sulfated fucose units released from the FCSs were analyzed via NMR and ESI-HRMS spectroscopies. The fucose units from each species presented extensive qualitative differences, but quantitative assessments of these units were hindered, mostly because of their extensive desulfation during the hydrolysis. The bottom-up analysis performed here has proved useful to explore the structure of FCS through a sum-of-the-parts approach in a qualitative manner. We further demonstrate that under specific acidification conditions particular fucose branches can be removed preferentially from FCS. Preparation of derivatives enriched with particular fucose branches could be useful for studies on "structure vs. biological function" of FCS. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Oncofetal Chondroitin Sulfate Glycosaminoglycans Are Key Players in Integrin Signaling and Tumor Cell Motility.

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Clausen, Thomas Mandel; Pereira, Marina Ayres; Al Nakouzi, Nader; Oo, Htoo Zarni; Agerbæk, Mette Ø; Lee, Sherry; Ørum-Madsen, Maj Sofie; Kristensen, Anders Riis; El-Naggar, Amal; Grandgenett, Paul M; Grem, Jean L; Hollingsworth, Michael A; Holst, Peter J; Theander, Thor; Sorensen, Poul H; Daugaard, Mads; Salanti, Ali

2016-12-01

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion, and anchorage-independent growth of tumor cells in vitro Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-β1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes β-1,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. The cancer-specific expression of ofCS aids in metastatic phenotypes and is a candidate target for therapy. Mol Cancer Res; 14(12); 1288-99. ©2016 AACR. ©2016 American Association for Cancer Research.

Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia

OpenAIRE

1989-01-01

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison o...

Synthesis of chondroitin sulfate CC and DD tetrasaccharides and interactions with 2H6 and LY111.

Science.gov (United States)

Matsushita, Kenya; Nakata, Tomomi; Takeda-Okuda, Naoko; Nadanaka, Satomi; Kitagawa, Hiroshi; Tamura, Jun-Ichi

2018-03-01

We synthesized the biotinylated chondroitin sulfate tetrasaccharides CS-CC [-3)βGalNAc6S(1-4)βGlcA(1-] 2 and CS-DD [-3)βGalNAc6S(1-4)βGlcA2S(1-] 2 which possess sulfate groups at O-6 of GalNAc and an additional sulfate group at O-2 of GlcA, respectively. We also analyzed interactions among CS-CC and CS-DD and the antibodies 2H6 and LY111, both of which are known to bind with CS-A, while CS-DD was shown for the first time to bind with both antibodies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Complex lipid trafficking in Niemann-Pick disease type C.

Science.gov (United States)

Vanier, Marie T

2015-01-01

Niemann-Pick disease type C (NPC) is an atypical lysosomal storage disease resulting from mutations in one of two genes, either NPC1 or NPC2. Although a neurovisceral disorder, it is above all a neurodegenerative disease in the vast majority of patients. Not an enzyme deficiency, it is currently conceived as a lipid trafficking disorder. Impaired egress of cholesterol from the late endosomal/lysosomal (LE/L) compartment is a specific and key element of the pathogenesis, but other lipids, more specially sphingolipids, are also involved, and there are indications for further abnormalities. The full function of the NPC1 and NPC2 proteins is still unclear. This review provides a reappraisal of lipid storage and lysosomal enzymes activities in tissues/cells from NPC patients and animal models. It summarizes the current knowledge on the NPC1 and NPC2 proteins and their function in transport of cholesterol within the late endosomal-lysosomal compartment, with emphasis on differences between systemic organs and the brain; it also discusses regulation by membrane lipids of the NPC2-mediated cholesterol trafficking, interplay between cholesterol and sphingomyelin, the metabolic origin of glycosphingolipids stored in brain, and the putative role of free sphingoid bases in pathogenesis. Brief mention is finally made of diseases affecting other genes that were very recently shown to impact the "NPC pathway".

Structural Insights into the Niemann-Pick C1 (NPC1)-Mediated Cholesterol Transfer and Ebola Infection.

Science.gov (United States)

Gong, Xin; Qian, Hongwu; Zhou, Xinhui; Wu, Jianping; Wan, Tao; Cao, Pingping; Huang, Weiyun; Zhao, Xin; Wang, Xudong; Wang, Peiyi; Shi, Yi; Gao, George F; Zhou, Qiang; Yan, Nieng

2016-06-02

Niemann-Pick disease type C (NPC) is associated with mutations in NPC1 and NPC2, whose gene products are key players in the endosomal/lysosomal egress of low-density lipoprotein-derived cholesterol. NPC1 is also the intracellular receptor for Ebola virus (EBOV). Here, we present a 4.4 Å structure of full-length human NPC1 and a low-resolution reconstruction of NPC1 in complex with the cleaved glycoprotein (GPcl) of EBOV, both determined by single-particle electron cryomicroscopy. NPC1 contains 13 transmembrane segments (TMs) and three distinct lumenal domains A (also designated NTD), C, and I. TMs 2-13 exhibit a typical resistance-nodulation-cell division fold, among which TMs 3-7 constitute the sterol-sensing domain conserved in several proteins involved in cholesterol metabolism and signaling. A trimeric EBOV-GPcl binds to one NPC1 monomer through the domain C. Our structural and biochemical characterizations provide an important framework for mechanistic understanding of NPC1-mediated intracellular cholesterol trafficking and Ebola virus infection. Copyright © 2016 Elsevier Inc. All rights reserved.

The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations.

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Scavenius, Carsten; Nikolajsen, Camilla Lund; Stenvang, Marcel; Thøgersen, Ida B; Wyrożemski, Łukasz; Wisniewski, Hans-Georg; Otzen, Daniel E; Sanggaard, Kristian W; Enghild, Jan J

2016-02-26

Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg(2+) or Mn(2+), but not Ca(2+), induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg(2+) found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidative Stress: A Pathogenic Mechanism for Niemann-Pick Type C Disease

Directory of Open Access Journals (Sweden)

Mary Carmen Vázquez

2012-01-01

Full Text Available Niemann-Pick type C (NPC disease is a neurovisceral atypical lipid storage disorder involving the accumulation of cholesterol and other lipids in the late endocytic pathway. The pathogenic mechanism that links the accumulation of intracellular cholesterol with cell death in NPC disease in both the CNS and the liver is currently unknown. Oxidative stress has been observed in the livers and brains of NPC mice and in different NPC cellular models. Moreover, there is evidence of an elevation of oxidative stress markers in the serumof NPC patients. Recent evidence strongly suggests that mitochondrial dysfunction plays an important role in NPC pathogenesis and that mitochondria could be a significant source of oxidative stress in this disease. In this context, the accumulation of vitamin E in the late endosomal/lysosomal compartments in NPC could lead to a potential decrease of its bioavailability and could be another possible cause of oxidative damage. Another possible source of reactive species in NPC is the diminished activity of different antioxidant enzymes. Moreover, because NPC is mainly caused by the accumulation of free cholesterol, oxidized cholesterol derivatives produced by oxidative stress may contribute to the pathogenesis of the disease.

Impaired Autophagy in the Lipid-Storage Disorder Niemann-Pick Type C1 Disease

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Sovan Sarkar

2013-12-01

Full Text Available Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1 disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-β-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease.

Wound healing and antibacterial activities of chondroitin sulfate- and acharan sulfate-reduced silver nanoparticles

International Nuclear Information System (INIS)

Im, A-Rang; Kim, Jee Young; Kim, Yeong Shik; Kim, Hyun-Seok; Cho, Seonho; Park, Youmie

2013-01-01

For topical applications in wound healing, silver nanoparticles (AgNPs) have attracted much attention as antibacterial agents. Herein, we describe a green-synthetic route for the production of biocompatible and crystalline AgNPs using two glycosaminoglycans, chondroitin sulfate (CS) and acharan sulfate (AS), as reducing agents. The synthetic approach avoids the use of toxic chemicals, and the yield of AgNPs formation is found to be 98.1% and 91.1% for the chondroitin sulfate-reduced silver nanoparticles (CS-AgNPs) and the acharan sulfate-reduced silver nanoparticles (AS-AgNPs), respectively. Nanoparticles with mostly spherical and amorphous shapes were observed, with an average diameter of 6.16 ± 2.26 nm for CS-AgNPs and 5.79 ± 3.10 nm for AS-AgNPs. Images of the CS-AgNPs obtained from atomic force microscopy revealed the self-assembled structure of CS was similar to a densely packed woven mat with AgNPs sprinkled on the CS. These nanoparticles were stable under cell culture conditions without any noticeable aggregation. An approximately 128-fold enhancement of the antibacterial activities of the AgNPs was observed against Enterobacter cloacae and Escherichia coli when compared to CS and AS alone. In addition, an in vivo animal model of wound healing activity was tested using mice that were subjected to deep incision wounds. In comparison to the controls, the ointments containing CS-AgNPs and AS-AgNPs stimulated wound closure under histological examination and accelerated the deposition of granulation tissue and collagen in the wound area. The wound healing activity of the ointments containing CS-AgNPs and AS-AgNPs are comparable to that of a commercial formulation of silver sulfadiazine even though the newly prepared ointments contain a lower silver concentration. Therefore, the newly prepared AgNPs demonstrate potential for use as an attractive biocompatible nanocomposite for topical applications in the treatment of wounds. (paper)

Wound healing and antibacterial activities of chondroitin sulfate- and acharan sulfate-reduced silver nanoparticles

Science.gov (United States)

Im, A.-Rang; Kim, Jee Young; Kim, Hyun-Seok; Cho, Seonho; Park, Youmie; Kim, Yeong Shik

2013-10-01

For topical applications in wound healing, silver nanoparticles (AgNPs) have attracted much attention as antibacterial agents. Herein, we describe a green-synthetic route for the production of biocompatible and crystalline AgNPs using two glycosaminoglycans, chondroitin sulfate (CS) and acharan sulfate (AS), as reducing agents. The synthetic approach avoids the use of toxic chemicals, and the yield of AgNPs formation is found to be 98.1% and 91.1% for the chondroitin sulfate-reduced silver nanoparticles (CS-AgNPs) and the acharan sulfate-reduced silver nanoparticles (AS-AgNPs), respectively. Nanoparticles with mostly spherical and amorphous shapes were observed, with an average diameter of 6.16 ± 2.26 nm for CS-AgNPs and 5.79 ± 3.10 nm for AS-AgNPs. Images of the CS-AgNPs obtained from atomic force microscopy revealed the self-assembled structure of CS was similar to a densely packed woven mat with AgNPs sprinkled on the CS. These nanoparticles were stable under cell culture conditions without any noticeable aggregation. An approximately 128-fold enhancement of the antibacterial activities of the AgNPs was observed against Enterobacter cloacae and Escherichia coli when compared to CS and AS alone. In addition, an in vivo animal model of wound healing activity was tested using mice that were subjected to deep incision wounds. In comparison to the controls, the ointments containing CS-AgNPs and AS-AgNPs stimulated wound closure under histological examination and accelerated the deposition of granulation tissue and collagen in the wound area. The wound healing activity of the ointments containing CS-AgNPs and AS-AgNPs are comparable to that of a commercial formulation of silver sulfadiazine even though the newly prepared ointments contain a lower silver concentration. Therefore, the newly prepared AgNPs demonstrate potential for use as an attractive biocompatible nanocomposite for topical applications in the treatment of wounds.

Ebola Viral Glycoprotein Bound to Its Endosomal Receptor Niemann-Pick C1.

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Wang, Han; Shi, Yi; Song, Jian; Qi, Jianxun; Lu, Guangwen; Yan, Jinghua; Gao, George F

2016-01-14

Filoviruses, including Ebola and Marburg, cause fatal hemorrhagic fever in humans and primates. Understanding how these viruses enter host cells could help to develop effective therapeutics. An endosomal protein, Niemann-Pick C1 (NPC1), has been identified as a necessary entry receptor for this process, and priming of the viral glycoprotein (GP) to a fusion-competent state is a prerequisite for NPC1 binding. Here, we have determined the crystal structure of the primed GP (GPcl) of Ebola virus bound to domain C of NPC1 (NPC1-C) at a resolution of 2.3 Å. NPC1-C utilizes two protruding loops to engage a hydrophobic cavity on head of GPcl. Upon enzymatic cleavage and NPC1-C binding, conformational change in the GPcl further affects the state of the internal fusion loop, triggering membrane fusion. Our data therefore provide structural insights into filovirus entry in the late endosome and the molecular basis for design of therapeutic inhibitors of viral entry. Copyright © 2016 Elsevier Inc. All rights reserved.

Pattern of chondroitin sulfate proteoglycan expression after ablation of the sensorimotor cortex of the neonatal and adult rat brain

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Dacić Sanja

2008-01-01

Full Text Available The central nervous system has a limited capacity for self-repair after damage. However, the neonatal brain has agreater capacity for recovery than the adult brain. These differences in the regenerative capability depend on local environmental factors and the maturational stage of growing axons. Among molecules which have both growth-promoting and growth-inhibiting activities is the heterogeneous class of chondroitin sulfate proteoglycans (CSPGs. In this paper, we investigated the chondroitin-4 and chondroitin-6 sulfate proteoglycan expression profile after left sensorimotor cortex ablation of the neonatal and adult rat brain. Immunohistochemical analysis revealed that compared to the normal uninjured cortex, lesion provoked up regulation of CSPGs showing a different pattern of expression in the neonatal vs. the adult brain. Punctuate and membrane-bound labeling was predominate after neonatal lesion, where as heavy deposition of staining in the extracellular matrix was observed after adult lesion. Heavy deposition of CSPG immunoreactivity around the lesionsite in adult rats, in contrast to a less CSPG-rich environment in neonatal rats, indicated that enhancement of the recovery process after neonatal injury is due to amore permissive environment.

Sulfation and cation effects on the conformational properties of the glycan backbone of chondroitin sulfate disaccharides.

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Faller, Christina E; Guvench, Olgun

2015-05-21

Chondroitin sulfate (CS) is one of several glycosaminoglycans that are major components of proteoglycans. A linear polymer consisting of repeats of the disaccharide -4GlcAβ1-3GalNAcβ1-, CS undergoes differential sulfation resulting in five unique sulfation patterns. Because of the dimer repeat, the CS glycosidic "backbone" has two distinct sets of conformational degrees of freedom defined by pairs of dihedral angles: (ϕ1, ψ1) about the β1-3 glycosidic linkage and (ϕ2, ψ2) about the β1-4 glycosidic linkage. Differential sulfation and the possibility of cation binding, combined with the conformational flexibility and biological diversity of CS, complicate experimental efforts to understand CS three-dimensional structures at atomic resolution. Therefore, all-atom explicit-solvent molecular dynamics simulations with Adaptive Biasing Force sampling of the CS backbone were applied to obtain high-resolution, high-precision free energies of CS disaccharides as a function of all possible backbone geometries. All 10 disaccharides (β1-3 vs β1-4 linkage × five different sulfation patterns) were studied; additionally, ion effects were investigated by considering each disaccharide in the presence of either neutralizing sodium or calcium cations. GlcAβ1-3GalNAc disaccharides have a single, broad, thermodynamically important free-energy minimum, whereas GalNAcβ1-4GlcA disaccharides have two such minima. Calcium cations but not sodium cations bind to the disaccharides, and binding is primarily to the GlcA -COO(-) moiety as opposed to sulfate groups. This binding alters the glycan backbone thermodynamics in instances where a calcium cation bound to -COO(-) can act to bridge and stabilize an interaction with an adjacent sulfate group, whereas, in the absence of this cation, the proximity of a sulfate group to -COO(-) results in two like charges being both desolvated and placed adjacent to each other and is found to be destabilizing. In addition to providing information

Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

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Farrugia, Brooke L; Whitelock, John M; O'Grady, Robert; Caterson, Bruce; Lord, Megan S

2016-02-01

The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells. © 2016 The Histochemical Society.

Oculomotor abnormalities in children with Niemann-Pick type C.

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Blundell, James; Frisson, Steven; Chakrapani, Anupam; Gissen, Paul; Hendriksz, Chris; Vijay, Suresh; Olson, Andrew

2018-02-01

Niemann-Pick type C (NP-C) is a rare recessive disorder associated with progressive supranuclear gaze palsy. Degeneration occurs initially for vertical saccades and later for horizontal saccades. There are studies of oculomotor degeneration in adult NP-C patients [1, 2] but no comparable studies in children. We used high-resolution video-based eye tracking to record monocular vertical and horizontal eye movements in 2 neurological NP-C patients (children with clinically observable oculomotor abnormalities) and 3 pre-neurological NP-C patients (children without clinically observable oculomotor abnormalities). Saccade onset latency, saccade peak velocity and saccade curvature were compared to healthy controls (N=77). NP-C patients had selective impairments of vertical saccade peak velocity and vertical saccade curvature, with slower peak velocities and greater curvature. Changes were more pronounced in neurological than pre-neurological patients, showing that these measures are sensitive to disease progress, but abnormal curvature and slowed downward saccades were present in both groups, showing that eye-tracking can register disease-related changes before these are evident in a clinical exam. Both slowing, curvature and the detailed characteristics of the curvature we observed are predicted by the detailed characteristics of RIMLF population codes. Onset latencies were not different from healthy controls. High-resolution video-based eye tracking is a promising sensitive and objective method to measure NP-C disease severity and neurological onset. It may also help evaluate responses to therapeutic interventions. Copyright © 2017. Published by Elsevier Inc.

Chondroitin sulfates and their binding molecules in the central nervous system.

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Djerbal, L; Lortat-Jacob, H; Kwok, Jcf

2017-06-01

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in the central nervous system (CNS) matrix. Its sulfation and epimerization patterns give rise to different forms of CS, which enables it to interact specifically and with a significant affinity with various signalling molecules in the matrix including growth factors, receptors and guidance molecules. These interactions control numerous biological and pathological processes, during development and in adulthood. In this review, we describe the specific interactions of different families of proteins involved in various physiological and cognitive mechanisms with CSs in CNS matrix. A better understanding of these interactions could promote a development of inhibitors to treat neurodegenerative diseases.

Iduronic Acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells

NARCIS (Netherlands)

Bartolini, B.; Thelin, M.A.; Svensson, L.; Ghiselli, G.; Kuppevelt, T.H. van; Malmstrom, A.; Maccarana, M.

2013-01-01

Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS

Highly sulfated hexasaccharide sequences isolated from chondroitin sulfate of shark fin cartilage: insights into the sugar sequences with bioactivities.

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Mizumoto, Shuji; Murakoshi, Saori; Kalayanamitra, Kittiwan; Deepa, Sarama Sathyaseelan; Fukui, Shigeyuki; Kongtawelert, Prachya; Yamada, Shuhei; Sugahara, Kazuyuki

2013-02-01

Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates and are linear polysaccharides consisting of variously sulfated repeating disaccharides, [-4GlcUAβ1-3GalNAcβ1-](n), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. CS chains containing D-disaccharide units [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)] are involved in the development of cerebellar Purkinje cells and neurite outgrowth-promoting activity through interaction with a neurotrophic factor, pleiotrophin, resulting in the regulation of signaling. In this study, to obtain further structural information on the CS chains containing d-disaccharide units involved in brain development, oligosaccharides containing D-units were isolated from a shark fin cartilage. Seven novel hexasaccharide sequences, ΔO-D-D, ΔA-D-D, ΔC-D-D, ΔE-A-D, ΔD-D-C, ΔE-D-D and ΔA-B-D, in addition to three previously reported sequences, ΔC-A-D, ΔC-D-C and ΔA-D-A, were isolated from a CS preparation of shark fin cartilage after exhaustive digestion with chondroitinase AC-I, which cannot act on the galactosaminidic linkages bound to D-units. The symbol Δ stands for a 4,5-unsaturated bond of uronic acids, whereas A, B, C, D, E and O represent [GlcUA-GalNAc(4-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(4-O-sulfate)], [GlcUA-GalNAc(6-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)], [GlcUA-GalNAc(4-O-, 6-O-sulfate)] and [GlcUA-GalNAc], respectively. In binding studies using an anti-CS monoclonal antibody, MO-225, the epitopes of which are involved in cerebellar development in mammals, novel epitope structures, ΔA-D-A, ΔA-D-D and ΔA-B-D, were revealed. Hexasaccharides containing two consecutive D-units or a B-unit will be useful for the structural and functional analyses of CS chains particularly in the neuroglycobiological fields.

Chondroitin sulfate A-adhering Plasmodium falciparum-infected erythrocytes express functionally important antibody epitopes shared by multiple variants

DEFF Research Database (Denmark)

Barfod, Lea; Dobrilovic, Tina; Magistrado, Pamela

2010-01-01

to chondroitin sulfate A in the intervillous space. Although interclonal variation of the var2csa gene is lower than that among var genes in general, VAR2CSA-specific Abs appear to target mainly polymorphic epitopes. This has raised doubts about the feasibility of VAR2CSA-based vaccines. We used eight human......) and recombinant VAR2CSA and interfered with IE and/or VAR2CSA binding to chondroitin sulfate A. Pair-wise mAb combinations were more inhibitory than single mAbs, and all of the mAbs together was the most efficient combination. Each mAb could opsonize IEs for phagocytosis, and a combination of the eight m...

Case Report: Genetic analysis and anesthetic management of a child with Niemann-Pick disease Type A [version 1; referees: 2 approved

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Priti G. Dalal

2015-12-01

Full Text Available A 14-month-old child, recently diagnosed with Niemann-Pick disease type A, presented for a laparoscopic placement of a gastrostomy tube under general anesthesia. The disease was confirmed and further characterized by genetic testing, which revealed evidence of the presence of two known pathogenic mutations in the SMPD1 gene, and enzyme studies showed a corresponding very low level of enzymatic activity of acidic sphingomyelinase. The anesthetic management involved strategies to manage an anticipated difficult intubation and avoid post-operative ventilation.

Preparation of Low Molecular Weight Chondroitin Sulfates, Screening of a High Anti-Complement Capacity of Low Molecular Weight Chondroitin Sulfate and Its Biological Activity Studies in Attenuating Osteoarthritis.

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Li, Lian; Li, Yan; Feng, Danyang; Xu, Linghua; Yin, Fengxin; Zang, Hengchang; Liu, Chunhui; Wang, Fengshan

2016-10-11

Chondroitin sulfate (CS) plays important roles in the complement system. However, the CS structure is complicated due to different sources and the number and positions of sulfate groups. The objective of this study was to prepare different low molecular weight chondroitin sulfates (LMWCSs) and to investigate the biological activity in anti-complement capacity. A series of LMWCSs was prepared from different sources and characterized by ultraviolet-visible (UV) spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) and nuclear magnetic resonance (NMR) spectroscopy. Hemolytic, anti-complement 3 deposition capacity and cell viability assays were carried out to investigate the biological activities in vitro. The results showed that LMWCS prepared from shark cartilage with the oxidative degradation method (LMWCS-S-O) had the best anti-complement capacity. LMWCS-S-O could inhibit the alternative pathway of the complement system and protect chondrocytes from cell death. The attenuating effect of LMWCS-S-O on Osteoarthritis (OA) was investigated by destabilization of the medial meniscus (DMM) model in vivo. Functional wind-up, histological and C5b-9 analyses were used to evaluate the treatment effect on the OA model. In vivo results showed that LMWCS-S-O could attenuate OA. LMWCS-S-O with a high content of ΔDi-2,6diS and ΔDi-6S could be used for attenuating OA through regulating the complement system.

Preparation of Low Molecular Weight Chondroitin Sulfates, Screening of a High Anti-Complement Capacity of Low Molecular Weight Chondroitin Sulfate and Its Biological Activity Studies in Attenuating Osteoarthritis

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Lian Li

2016-10-01

Full Text Available Chondroitin sulfate (CS plays important roles in the complement system. However, the CS structure is complicated due to different sources and the number and positions of sulfate groups. The objective of this study was to prepare different low molecular weight chondroitin sulfates (LMWCSs and to investigate the biological activity in anti-complement capacity. A series of LMWCSs was prepared from different sources and characterized by ultraviolet-visible (UV spectroscopy, high-performance liquid chromatography (HPLC, size exclusion chromatography-multiangle laser light scattering (SEC-MALLS and nuclear magnetic resonance (NMR spectroscopy. Hemolytic, anti-complement 3 deposition capacity and cell viability assays were carried out to investigate the biological activities in vitro. The results showed that LMWCS prepared from shark cartilage with the oxidative degradation method (LMWCS-S-O had the best anti-complement capacity. LMWCS-S-O could inhibit the alternative pathway of the complement system and protect chondrocytes from cell death. The attenuating effect of LMWCS-S-O on Osteoarthritis (OA was investigated by destabilization of the medial meniscus (DMM model in vivo. Functional wind-up, histological and C5b-9 analyses were used to evaluate the treatment effect on the OA model. In vivo results showed that LMWCS-S-O could attenuate OA. LMWCS-S-O with a high content of ΔDi-2,6diS and ΔDi-6S could be used for attenuating OA through regulating the complement system.

Quantitative analysis of glycosaminoglycans, chondroitin/dermatan sulfate, hyaluronic acid, heparan sulfate, and keratan sulfate by liquid chromatography-electrospray ionization-tandem mass spectrometry.

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Osago, Harumi; Shibata, Tomoko; Hara, Nobumasa; Kuwata, Suguru; Kono, Michihaya; Uchio, Yuji; Tsuchiya, Mikako

2014-12-15

We developed a method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive "GAGomic" analysis of biological tissues. Copyright © 2014 Elsevier Inc. All rights reserved.

Chondroitin Sulfate Inhibits Monocyte Chemoattractant Protein-1 Release From 3T3-L1 Adipocytes: A New Treatment Opportunity for Obesity-Related Inflammation?

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Thomas V Stabler

2017-08-01

Full Text Available Monocyte chemoattractant protein-1 (MCP-1 overproduction from inflamed adipose tissue is a major contributor to obesity-related metabolic syndromes. 3T3-L1 embryonic fibroblasts were cultured and differentiated into adipocytes using an established protocol. Adipocytes were treated with lipopolysaccharide (LPS to induce inflammation and thus MCP-1 release. At the same time, varying concentrations of chondroitin sulfate (CS were added in a physiologically relevant range (10-200 µg/mL to determine its impact on MCP-1 release. Chondroitin sulfate, a natural glycosaminoglycan of connective tissue including the cartilage extracellular matrix, was chosen on the basis of our previous studies demonstrating its anti-inflammatory effect on macrophages. Because the main action of MCP-1 is to induce monocyte migration, cultured THP-1 monocytes were used to test whether CS at the highest physiologically relevant concentration could inhibit cell migration induced by human recombinant MCP-1. Chondroitin sulfate (100-200 µg/mL inhibited MCP-1 release from inflamed adipocytes in a dose-dependent manner ( P  < .01, 95% confidence interval [CI]: −5.89 to −3.858 at 100 µg/mL and P  < .001, 95% CI: −6.028 to −3.996 at 200 µg/mL but had no effect on MCP-1–driven chemotaxis of THP-1 monocytes. In summary, CS could be expected to reduce macrophage infiltration into adipose tissue by reduction in adipocyte expression and release of MCP-1 and as such might reduce adipose tissue inflammation in response to pro-inflammatory stimuli such as LPS, now increasingly recognized to be relevant in vivo.

Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias

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Shuji Mizumoto

2015-01-01

Full Text Available Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

Impaired autophagy in the lipid-storage disorder Niemann-Pick type C1 disease.

Science.gov (United States)

Sarkar, Sovan; Carroll, Bernadette; Buganim, Yosef; Maetzel, Dorothea; Ng, Alex H M; Cassady, John P; Cohen, Malkiel A; Chakraborty, Souvik; Wang, Haoyi; Spooner, Eric; Ploegh, Hidde; Gsponer, Joerg; Korolchuk, Viktor I; Jaenisch, Rudolf

2013-12-12

Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-β-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration

DEFF Research Database (Denmark)

Agerbaek, Mette Ø; Bento Ayres Pereira, Marina Maria; Clausen, Thomas M

2017-01-01

in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored...

cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

DEFF Research Database (Denmark)

Wu, R R; Couchman, J R

1997-01-01

Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now....... The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser...

Treatment of cataplexy in Niemann-Pick disease type C with the use of miglustat.

Science.gov (United States)

Zarowski, Marcin; Steinborn, Barbara; Gurda, Barbara; Dvorakova, Lenka; Vlaskova, Hana; Kothare, Sanjeev V

2011-01-01

Cataplexy is the sudden muscle weakness brought on by strong emotions, particularly joking, laughter, or anger. Cataplexy may involve only certain group of muscles or the entire voluntary musculature. In rare cases, symptoms of cataplexy can be seen during the course of some inherited diseases (Niemann-Pick type C (NPC), Prader-Willi syndrome, myotonic dystrophy, Norrie disease). We report the successful use of miglustat, a reversible inhibitor of the enzyme glucosylceramide synthase, approved for use in Gaucher's disease, and which catalyses the first step in the biosynthesis of most glycosphingolipid, in a boy with NPC with cataplexy. A 9-year-old boy was admitted for assessments of frequent "drop attacks" while laughing. The filipin fluorescence tests of cultured skin fibroblasts revealed massive accumulation of unesterified cholesterol, confirming the diagnosis of NPC disease. Molecular studies confirmed the diagnosis of NPC too. After approval from the bioethics committee, miglustat was initiated on the child at 100mg three times a day. Cataplectic attacks disappeared completely after 6 months on treatment, and patient continues to be in remission from the cataplectic attacks at 16 months follow-up. There was no further progression of neurological signs or symptoms or splenomegaly, with some improvement in cognitive function as well as social, affective and attention problems, up-gaze, and gait. Miglustat was well tolerated with no side effects observed. In summary, this is the first report of miglustat treatment of cataplexy in NPC. Long-term follow-up for continuing efficacy and tolerability in a larger cohort with NPC is needed to substantiate our observation. © 2010 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

A thermo-responsive and photo-polymerizable chondroitin sulfate-based hydrogel for 3D printing applications

NARCIS (Netherlands)

Abbadessa, A.; Blokzijl, M. M.; Mouser, V. H. M.; Marica, P.; Malda, J.; Hennink, W. E.; Vermonden, T.

2016-01-01

The aim ofthis study was to design a hydrogel system based on methacrylated chondroitin sulfate (CSMA) and a thermo-sensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate)-polyethylene glycol triblock copolymer (M15P10) as a suitable material for additive manufacturing of scaffolds. CSMA

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. (II.) Seven compounds containing 2 or 3 sulfate residues.

NARCIS (Netherlands)

Vliegenthart, J.F.G.; Waard, P. de; Harada, T.; Sugahara, K.

1992-01-01

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols were obtained from the carbohydrate-protein linkage region and six of them contain 0 or 1

A thermo-responsive and photo-polymerizable chondroitin sulfate-based hydrogel for 3D printing applications

NARCIS (Netherlands)

Abbadessa, A|info:eu-repo/dai/nl/369480376; Blokzijl, M M; Mouser, V H M; Marica, P; Malda, J|info:eu-repo/dai/nl/412461099; Hennink, W E|info:eu-repo/dai/nl/070880409; Vermonden, T|info:eu-repo/dai/nl/275124517

2016-01-01

The aim of this study was to design a hydrogel system based on methacrylated chondroitin sulfate (CSMA) and a thermo-sensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate)-polyethylene glycol triblock copolymer (M15P10) as a suitable material for additive manufacturing of scaffolds. CSMA

Novel single-chain antibody GD3A10 defines a chondroitin sulfate biomarker for ovarian cancer

NARCIS (Netherlands)

Vallen, M.J.E.; Tilborg, A.G. van; Tesselaar, M.H.; Dam, G.B. ten; Bulten, J.; Kuppevelt, T.H. van; Massuger, L.F.A.G.

2014-01-01

AIMS: Ovarian cancer has the highest case-to-fatality-index of all gynecological cancers. In this study, tumor-related alterations in the extracellular matrix, especially regarding chondroitin sulfate glycosaminoglycans, are proposed as a novel biomarker in ovarian cancer. MATERIALS & METHODS: Phage

Preparation and antifouling property of polyurethane film modified by chondroitin sulfate

Science.gov (United States)

Yuan, Huihui; Xue, Jing; Qian, Bin; Chen, Huaying; Zhu, Yonggang; Lan, Minbo

2017-02-01

An antifouling polyurethane film modified by chondroitin sulfate (PU-CS) was prepared by chemical grafting with N-Boc-1,3-propanediamine as a spacer. The different mass fraction of N-Boc-1,3-propanediamine was investigated to obtain PU-CS films with different CS grafting density. The surface properties of PU-CS films were comprehensively characterized. Proteins adsorption and glycosaminoglycans adhesion on films were evaluated. Moreover, inorganic salt deposition on film with highest CS grafting density (3.70 μg/cm2) was briefly investigated. The results showed that the increase of CS grafting density improved not only the hydrophilicity but the antifouling performance of films. The best antifouling film reduced the adsorption of fibrinogen (BFG), human serum albumin (HSA) and lysozyme (LYS) by 81.4%, 95.0% and 76.5%, respectively, and the adhesion of chondroitin (CS), heparin (HP) and hyaluronic acid (HA) by 70.6%, 87.4% and 81.3%, respectively. In addition, the co-adsorption of proteins and glycosaminoglycans reduced up to 86.9% and 75.5%, respectively. Changes in inorganic salt deposition after co-adsorption of proteins and glycosaminoglycans on PU-CS(3) suggested that the proteins promoted the inorganic salt deposition, while glycosaminoglycans inhibited the crystal growth. The negatively charged polysaccharides might promote the generation of smaller crystals which could be conducive to provide theoretical and practical guide to develop novel urinary stents with significant anti-encrustation properties.

An Injectable Enzymatically Crosslinked Carboxymethylated Pullulan/Chondroitin Sulfate Hydrogel for Cartilage Tissue Engineering

Science.gov (United States)

Chen, Feng; Yu, Songrui; Liu, Bing; Ni, Yunzhou; Yu, Chunyang; Su, Yue; Zhu, Xinyuan; Yu, Xiaowei; Zhou, Yongfeng; Yan, Deyue

2016-01-01

In this study, an enzymatically cross-linked injectable and biodegradable hydrogel system comprising carboxymethyl pullulan-tyramine (CMP-TA) and chondroitin sulfate-tyramine (CS-TA) conjugates was successfully developed under physiological conditions in the presence of both horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) for cartilage tissue engineering (CTTE). The HRP crosslinking method makes this injectable system feasible, minimally invasive and easily translatable for regenerative medicine applications. The physicochemical properties of the mechanically stable hydrogel system can be modulated by varying the weight ratio and concentration of polymer as well as the concentrations of crosslinking reagents. Additionally, the cellular behaviour of porcine auricular chondrocytes encapsulated into CMP-TA/CS-TA hydrogels demonstrates that the hydrogel system has a good cyto-compatibility. Specifically, compared to the CMP-TA hydrogel, these CMP-TA/CS-TA composite hydrogels have enhanced cell proliferation and increased cartilaginous ECM deposition, which significantly facilitate chondrogenesis. Furthermore, histological analysis indicates that the hydrogel system exhibits acceptable tissue compatibility by using a mouse subcutaneous implantation model. Overall, the novel injectable pullulan/chondroitin sulfate composite hydrogels presented here are expected to be useful biomaterial scaffold for regenerating cartilage tissue.

Inhibition of chondroitin sulfate glycosaminoglycans incorporation affected odontoblast differentiation in cultured embryonic mouse molars.

Science.gov (United States)

Liu, Lipei; Chen, Weiting; Li, Lefeng; Xu, Fangfang; Jiang, Beizhan

2017-12-01

Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without β-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by β-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that β-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.

Phosphatidylcholine nanovesicles coated with chitosan or chondroitin sulfate as novel devices for bacteriocin delivery

Science.gov (United States)

da Silva, Indjara Mallmann; Boelter, Juliana Ferreira; da Silveira, Nádya Pesce; Brandelli, Adriano

2014-07-01

There is increased interest on the use of natural antimicrobial peptides in biomedicine and food preservation technologies. In this study, the antimicrobial activity of nisin encapsulated into nanovesicles containing polyanionic polysaccharides was investigated. Nisin was encapsulated in phosphatidylcholine (PC) liposomes containing chitosan or chondroitin sulfate by the thin-film hydration method and tested for antimicrobial activity against Listeria spp. The mean particle size of PC liposomes was 145 nm and varied to 210 and 134 nm with the incorporation of chitosan and chondroitin sulfate, respectively. Nisin-containing nanovesicles with and without incorporation of polysaccharides had a zeta potential values around -20 mV, showing mostly spherical structures when observed by transmission electron microscopy. Encapsulated nisin had similar efficiency as free nisin in inhibiting Listeria spp. isolated from bovine carcass, and greater efficiency in inhibiting Listeria monocytogenes. The formulation containing chitosan was more stable and more efficient in inhibiting L. monocytogenes when compared to the other nanovesicles tested. After 24 h, the viable cell counts were 2 log lower as compared with the other treatments and 7 log comparing to controls.

Niemann-Pick C1-deficient mice lacking sterol O-acyltransferase 2 have less hepatic cholesterol entrapment and improved liver function.

Science.gov (United States)

Lopez, Adam M; Jones, Ryan Dale; Repa, Joyce J; Turley, Stephen D

2018-06-07

Cholesteryl esters are generated at multiple sites in the body by sterol O-acyltransferase 1 (SOAT1) or sterol O-acyltransferase 2 (SOAT2) in various cell types, and lecithin cholesterol acyltransferase (LCAT) in plasma. Esterified cholesterol (EC) and triacylglycerol (TAG) contained in lipoproteins cleared from the circulation via receptor-mediated or bulk-phase endocytosis are hydrolyzed by lysosomal acid lipase (LAL) within the late endosomal/lysosomal (E/L) compartment. Then, through the successive actions of Niemann-Pick C2 (NPC2) and Niemann-Pick C1 (NPC1), unesterified cholesterol (UC) is exported from the E/L compartment to the cytosol. Mutations in either NPC1 or NPC2 lead to continuing entrapment of UC in all organs, resulting in multisystem disease which includes hepatic dysfunction and in some cases liver failure. These studies investigated primarily whether elimination of SOAT2 in NPC1-deficient mice impacted hepatic UC sequestration, inflammation, and transaminase activities. Measurements were made in 7 wk-old mice fed a low-cholesterol chow diet or one enriched with cholesterol starting 2 wk before study. In the chow-fed mice, NPC1:SOAT2 double knockouts, compared to their littermates lacking only NPC1, had 20% less liver mass, 28% lower hepatic UC concentrations, and plasma ALT and AST activities that were decreased by 48% and 36%, respectively. mRNA expression levels for several markers of inflammation were all significantly lower in the NPC1 mutants lacking SOAT2. The existence of a new class of potent and selective SOAT2 inhibitors provides an opportunity for exploring if suppression of this enzyme could potentially become an adjunctive therapy for liver disease in NPC1 deficiency.

Chondroitin sulfate template-mediated biomimetic synthesis of nano-flake hydroxyapatite

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He, Dan; Xiao, Xiufeng; Liu, Fang; Liu, Rongfang

2008-11-01

By Ca(NO 3) 2·4H 2O and (NH 4) 3PO 4·3H 2O as reagents and chondroitin sulfate (ChS) as a template, nano-flake hydroxyapatite (HA) is synthesized using a biomimetic method according to the biomineralization theory. HA crystals obtained are characterized in crystalline phase, microstructure, chemical composition and morphology by X-ray diffraction (XRD), Fourier transform infrared spectroscope (FTIR), transmission electron microscopy (TEM) and elemental analysis respectively. UV-vis spectrum is adopted to investigate interactions between functional groups ChS and HA. The results show that HA crystal nucleation and growth take place in chemical interactions between HA crystals and ChS as a template. And elemental analysis indicates that obtained HA contains a small amount of ChS. Furthermore, ChS concentration significantly affects the morphology of HA crystals. Staple-fiber-like HA crystals can be obtained at a low concentration in ChS, and flake-like HA crystals synthesized at a high concentration (≥0.5 wt.%) of ChS as a template.

Chondroitin sulfate template-mediated biomimetic synthesis of nano-flake hydroxyapatite

Energy Technology Data Exchange (ETDEWEB)

He Dan; Xiao Xiufeng; Liu Fang [College of Chemistry and Materials Science, Fujian Normal University, Fuzhou 350007 (China); Liu Rongfang [College of Chemistry and Materials Science, Fujian Normal University, Fuzhou 350007 (China)], E-mail: rfliu@vip.sina.com

2008-11-15

By Ca(NO{sub 3}){sub 2}.4H{sub 2}O and (NH{sub 4}){sub 3}PO{sub 4}.3H{sub 2}O as reagents and chondroitin sulfate (ChS) as a template, nano-flake hydroxyapatite (HA) is synthesized using a biomimetic method according to the biomineralization theory. HA crystals obtained are characterized in crystalline phase, microstructure, chemical composition and morphology by X-ray diffraction (XRD), Fourier transform infrared spectroscope (FTIR), transmission electron microscopy (TEM) and elemental analysis respectively. UV-vis spectrum is adopted to investigate interactions between functional groups ChS and HA. The results show that HA crystal nucleation and growth take place in chemical interactions between HA crystals and ChS as a template. And elemental analysis indicates that obtained HA contains a small amount of ChS. Furthermore, ChS concentration significantly affects the morphology of HA crystals. Staple-fiber-like HA crystals can be obtained at a low concentration in ChS, and flake-like HA crystals synthesized at a high concentration ({>=}0.5 wt.%) of ChS as a template.

Niemann-Pick disease Type C - Sea-blue histiocytosis: Phenotypic and imaging observations and mini review

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Praveen K

2007-01-01

Full Text Available We present a report on an 18-year-old boy with Niemann-Pick disease Type C (NP-C who presented with progressive decline in scholastic performance since 9 years of age. At 12 years, he developed abnormal behavior and after 2 years had insidious onset, progressive gait ataxia and dysarthria followed by dystonia of the right upper extremity, excessive drooling, dysphagia and nasal regurgitation. He had coarse facies, depressed nasal bridge, high arched palate, crowded teeth, splenomegaly and peculiar facial grin. In addition, impaired vertical saccadic and pursuit eye movements, brisk muscle stretch reflexes and limb and gait ataxia were observed. He had a low IQ of 47 on Binet-Kamat test. The ultrasound examination of the abdomen confirmed the presence of moderate splenomegaly. Magnetic resonance imaging brain showed symmetrical leucoencephalopathy and mild cerebellar atrophy. Bone marrow aspiration showed numerous foamy macrophages and sea-blue histiocytes suggesting the diagnosis of NP-C.

Reducing GBA2 Activity Ameliorates Neuropathology in Niemann-Pick Type C Mice.

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André R A Marques

Full Text Available The enzyme glucocerebrosidase (GBA hydrolyses glucosylceramide (GlcCer in lysosomes. Markedly reduced GBA activity is associated with severe manifestations of Gaucher disease including neurological involvement. Mutations in the GBA gene have recently also been identified as major genetic risk factor for Parkinsonism. Disturbed metabolism of GlcCer may therefore play a role in neuropathology. Besides lysosomal GBA, cells also contain a non-lysosomal glucosylceramidase (GBA2. Given that the two β-glucosidases share substrates, we speculated that over-activity of GBA2 during severe GBA impairment might influence neuropathology. This hypothesis was studied in Niemann-Pick type C (Npc1-/- mice showing secondary deficiency in GBA in various tissues. Here we report that GBA2 activity is indeed increased in the brain of Npc1-/- mice. We found that GBA2 is particularly abundant in Purkinje cells (PCs, one of the most affected neuronal populations in NPC disease. Inhibiting GBA2 in Npc1-/- mice with a brain-permeable low nanomolar inhibitor significantly improved motor coordination and extended lifespan in the absence of correction in cholesterol and ganglioside abnormalities. This trend was recapitulated, although not to full extent, by introducing a genetic loss of GBA2 in Npc1-/- mice. Our findings point to GBA2 activity as therapeutic target in NPC.

Modeling Niemann Pick type C1 using human embryonic and induced pluripotent stem cells.

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Ordoñez, M Paulina; Steele, John W

2017-02-01

Data generated in Niemann Pick type C1 (NPC1) human embryonic and human induced pluripotent stem cell derived neurons complement on-going studies in animal models and provide the first example, in disease-relevant human cells, of processes that underlie preferential neuronal defects in a NPC1. Our work and that of other investigators in human neurons derived from stem cells highlight the importance of performing rigorous mechanistic studies in relevant cell types to guide drug discovery and therapeutic development, alongside of existing animal models. Through the use of human stem cell-derived models of disease, we can identify and discover or repurpose drugs that revert early events that lead to neuronal failure in NPC1. Together with the study of disease pathogenesis and efficacy of therapies in animal models, these strategies will fulfill the promise of stem cell technology in the development of new treatments for human diseases. This article is part of a Special Issue entitled SI: Exploiting human neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

Developmental and functional significance of the CSF-1 proteoglycan chondroitin sulfate chain

OpenAIRE

Nandi, Sayan; Akhter, Mohammed P.; Seifert, Mark F.; Dai, Xu-Ming; Stanley, E. Richard

2006-01-01

The primary macrophage growth factor, colony-stimulating factor-1 (CSF-1), is homodimeric and exists in 3 biologically active isoforms: a membrane-spanning, cell-surface glycoprotein (csCSF-1) and secreted glycoprotein (sgCSF-1) and proteoglycan (spCSF-1) isoforms. To investigate the in vivo role of the chondroitin sulfate glycosaminoglycan (GAG) chain of spCSF-1, we created mice that exclusively express, in a normal tissue-specific and developmental manner, either the secreted precursor of s...

Visualization of liver on bone scintigraphy during treatment with iron chondroitin sulfate colloid

International Nuclear Information System (INIS)

Tanaka, Shigeko; Sawa, Hisashi; Fukuda, Teruo

1983-01-01

In order to see its probable cause in the incidental liver delineation on the bone scan with sup(99m)Tc-MDP, patients and experimental rabbits were intravenously injected with Blutal (iron lhondroitin sulfate) varicus times following the intravenous injection of sup(99m)Tc-MDP. The liver images were obtained during the early periods. An injection of Blutal-sup(99m)Tc pertechnetate mixture did not result in any appreciable hepatic delineation. Stannous chloride in the MDP kit could have enticed formation of radiocolloids in the presence of iron chondroitin sulfate. Diagnostic problems can be evaded by carrefully planning the bone scintigraphy in relation to the Blutal administration. (author)

Sensitive detection of oversulfated chondroitin sulfate in heparin sodium or crude heparin with a colorimetric microplate based assay.

Science.gov (United States)

Sommers, Cynthia D; Mans, Daniel J; Mecker, Laura C; Keire, David A

2011-05-01

In this work we describe a 96-well microplate assay for oversulfated chondroitin sulfate A (OSCS) in heparin, based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N'-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. The assay takes advantage of several unique properties of heparin, OSCS, and LPTP, including OSCS inhibition of heparinase I and II activity, the molecular weight dependence of heparin-LPTP spectral shifts, and the distinct association of heparin fragments and OSCS to LPTP. These factors combine to enable detection of the presence of 0.003% w/w spiked OSCS in 10 μg of heparin sodium active pharmaceutical ingredient (API) using a plate reader and with visual detection to 0.1% levels. The same detection limit for OSCS was observed in the presence of 10% levels of dermatan sulfate (DS) or chondroitin sulfate A (CSA) impurities. In addition, we surveyed a selection of crude heparin samples received by the agency in 2008 and 2009 to determine average and extreme DS, CSA, and galactosamine weight percent levels. In the presence of these impurities and the variable heparin content in the crude heparin samples, spiked OSCS was reliably detected to the 0.1% w/w level using a plate reader. Finally, authentically OSCS contaminated heparin sodium API and crude samples were distinguished visually by color from control samples using the LPTP/heparinase test.

Hypochlorite-mediated fragmentation of hyaluronan, chondroitin sulfates, and related N-acetyl glycosamines

DEFF Research Database (Denmark)

Rees, Martin D; Hawkins, Clare Louise; Davies, Michael Jonathan

2003-01-01

Myeloperoxidase released from activated phagocytes reacts with H(2)O(2) in the presence of chloride ions to give hypochlorous acid. This oxidant has been implicated in the fragmentation of glycosaminoglycans, such as hyaluronan and chondroitin sulfates. In this study it is shown that reaction...... processes. In the case of glycosaminoglycan-derived amidyl radicals, evidence has been obtained in studies with model glycosides that these radicals undergo rapid intramolecular abstraction reactions to give carbon-centered radicals at C-2 on the N-acetyl glycosamine rings (via a 1,2-hydrogen atom shift......) and at C-4 on the neighboring uronic acid residues (via 1,5-hydrogen atom shifts). The C-4 carbon-centered radicals, and analogous species derived from model glycosides, undergo pH-independent beta-scission reactions that result in glycosidic bond cleavage. With N-acetyl glucosamine C-1 alkyl glycosides...

Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycans Are Targeted by Bleomycin in Cancer Cells.

Science.gov (United States)

Li, Xiulian; Lan, Ying; He, Yanli; Liu, Yong; Luo, Heng; Yu, Haibo; Song, Ni; Ren, Sumei; Liu, Tianwei; Hao, Cui; Guo, Yunliang; Zhang, Lijuan

2017-01-01

Bleomycin is a clinically used anti-cancer drug that produces DNA breaks once inside of cells. However, bleomycin is a positively charged molecule and cannot get inside of cells by free diffusion. We previously reported that the cell surface negatively charged glycosaminoglycans (GAGs) may be involved in the cellular uptake of bleomycin. We also observed that a class of positively charged small molecules has Golgi localization once inside of the cells. We therefore hypothesized that bleomycin might perturb Golgi-operated GAG biosynthesis. We used stable isotope labeling coupled with LC/MS analysis of GAG disaccharides simultaneously from bleomycin-treated and non-treated cancer cells. To further understand the cytotoxicity of bleomycin and its relationship to GAGs, we used sodium chlorate to inhibit GAG sulfation and commercially available GAGs to compete for cell surface GAG/bleomycin interactions in seven cell lines including CHO745 defective in both heparan sulfate and chondroitin sulfate biosynthesis. we discovered that heparan sulfate GAG was significantly undersulfated and the quantity and disaccharide compositions of GAGs were changed in bleomycin-treated cells in a concentration- and time-dependent manner. We revealed that bleomycin-induced cytotoxicity was directly related to cell surface GAGs. GAGs were targeted by bleomycin both at cell surface and at Golgi. Thus, GAGs might be the biological relevant molecules that might be related to the bleomycin-induced fibrosis in certain cancer patients, a severe side effect with largely unknown molecular mechanism. © 2017 The Author(s). Published by S. Karger AG, Basel.

Equivalence of a single dose (1200 mg) compared to a three-time a day dose (400 mg) of chondroitin 4&6 sulfate in patients with knee osteoarthritis. Results of a randomized double blind placebo controlled study.

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Zegels, B; Crozes, P; Uebelhart, D; Bruyère, O; Reginster, J Y

2013-01-01

Evaluation of the efficacy and safety of a single oral dose of a 1200 mg sachet of chondroitin 4&6 sulfate (CS 1200) vs three daily capsules of chondroitin 4&6 sulfate 400 mg (CS 3*400) (equivalence study) and vs placebo (superiority study) during 3 months, in patients with knee osteoarthritis (OA). Comparative, double-blind, randomized, multicenter study, including 353 patients of both genders over 45 years with knee OA. Minimum inclusion criteria were a Lequesne index (LI) ≥ 7 and pain ≥ 40 mm on a visual analogue scale (VAS). LI and VAS were assessed at baseline and after 1-3 months. Equivalence between CS was tested using the per-protocol procedure and superiority of CS vs placebo was tested using an intent-to-treat procedure. After 3 months of follow-up, no significant difference was demonstrated between the oral daily single dose of CS 1200 formulation and the three daily capsules of CS 400. Patients treated with CS 1200 or CS 3*400 were significantly improved compared to placebo after 3 months of follow-up in terms of LI (security and tolerability was observed between the three groups. This study suggests that a daily administration of an oral sachet of 1200 mg of chondroitin 4&6 sulfate allows a significant clinical improvement compared to a placebo, and a similar improvement when compared to a regimen of three daily capsules of 400 mg of the same active ingredient. Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Facile analysis of contents and compositions of the chondroitin sulfate/dermatan sulfate hybrid chain in shark and ray tissues.

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Takeda, Naoko; Horai, Sawako; Tamura, Jun-ichi

2016-04-07

The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from specific tissues of several kinds of sharks and rays. The contents and sulfation patterns of the CS/DS hybrid chain were precisely analyzed by digestion with chondroitinases ABC and AC. All samples predominantly contained the A- and C-units. Furthermore, all samples characteristically contained the D-unit. Species-specific differences were observed in the contents of the CS/DS hybrid chain, which were the highest in Mako and Blue sharks and Sharpspine skates, but were lower in Hammerhead sharks. Marked differences were observed in the ratio of the C-unit/A-unit between sharks and rays. The contents of the CS/DS hybrid chain and the ratio of the C-unit/A-unit may be related to an oxidative stress-decreasing ability. Copyright © 2016 Elsevier Ltd. All rights reserved.

Kinetics of corrosion inhibition of aluminum in acidic media by water-soluble natural polymeric chondroitin-4-sulfate as anionic polyelectrolyte inhibitor.

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Hassan, Refat M; Ibrahim, Samia M; Takagi, Hideo D; Sayed, Suzan A

2018-07-15

Corrosion inhibition of aluminum (Al) in hydrochloric acid by anionic polyelectrolyte chondroitin-4-sulfate (CS) polysaccharide has been studied using both gasometrical and weight-loss techniques. The results drawn from these two techniques are comparable and exhibit negligible differences. The inhibition efficiency was found to increase with increasing the inhibitor concentration and decreased with increasing temperature. The inhibition action of CS on Al metal surface was found to obey both of Langmuir and Freundlich isotherms. The factors affecting the corrosion rates such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and the temperature were examined. The kinetic parameters were evaluated and a suitable corrosion mechanism consistent with the results obtained is discussed. Copyright © 2018. Published by Elsevier Ltd.

The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains

DEFF Research Database (Denmark)

Dahlbäck, Madeleine; Jørgensen, Lars M; Nielsen, Morten A

2011-01-01

Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated...

Hexuronic Acid Stereochemistry Determination in Chondroitin Sulfate Glycosaminoglycan Oligosaccharides by Electron Detachment Dissociation

Science.gov (United States)

Leach, Franklin E.; Ly, Mellisa; Laremore, Tatiana N.; Wolff, Jeremy J.; Perlow, Jacob; Linhardt, Robert J.; Amster, I. Jonathan

2012-09-01

Electron detachment dissociation (EDD) has previously provided stereo-specific product ions that allow for the assignment of the acidic C-5stereochemistry in heparan sulfate glycosaminoglycans (GAGs), but application of the same methodology to an epimer pair in the chondroitin sulfate glycoform class does not provide the same result. A series of experiments have been conducted in which glycosaminoglycan precursor ions are independently activated by electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD) to assign the stereochemistry in chondroitin sulfate (CS) epimers and investigate the mechanisms for product ion formation during EDD in CS glycoforms. This approach allows for the assignment of electronic excitation products formed by EID and detachment products to radical pathways in NETD, both of which occur simultaneously during EDD. The uronic acid stereochemistry in electron detachment spectra produces intensity differences when assigned glycosidic and cross-ring cleavages are compared. The variations in the intensities of the doubly deprotonated 0,2X3 and Y3 ions have been shown to be indicative of CS-A/DS composition during the CID of binary mixtures. These ions can provide insight into the uronic acid composition of binary mixtures in EDD, but the relative abundances, although reproducible, are low compared with those in a CID spectrum acquired on an ion trap. The application of principal component analysis (PCA) presents a multivariate approach to determining the uronic acid stereochemistry spectra of these GAGs by taking advantage of the reproducible peak distributions produced by electron detachment.

Olfactory deficits in Niemann-Pick type C1 (NPC1 disease.

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Marina Hovakimyan

Full Text Available BACKGROUND: Niemann-Pick type C disease (NPC is a rare autosomal recessive lipid storage disease characterized by progressive neurodegeneration. As only a few studies have been conducted on the impact of NPC on sensory systems, we used a mutant mouse model (NPC1(-/- to examine the effects of this disorder to morphologically distinct regions of the olfactory system, namely the olfactory epithelium (OE and olfactory bulb (OB. METHODOLOGY/PRINCIPAL FINDINGS: For structural and functional analysis immunohistochemistry, electron microscopy, western blotting, and electrophysiology have been applied. For histochemistry and western blotting, we used antibodies against a series of neuronal and glia marker proteins, as well as macrophage markers. NPC1(-/- animals present myelin-like lysosomal deposits in virtually all types of cells of the peripheral and central olfactory system. Especially supporting cells of the OE and central glia cells are affected, resulting in pronounced astrocytosis and microgliosis in the OB and other olfactory cortices. Up-regulation of Galectin-3, Cathepsin D and GFAP in the cortical layers of the OB underlines the critical role and location of the OB as a possible entrance gate for noxious substances. Unmyelinated olfactory afferents of the lamina propria seem less affected than ensheathing cells. Supporting the structural findings, electro-olfactometry of the olfactory mucosa suggests that NPC1(-/- animals exhibit olfactory and trigeminal deficits. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a pronounced neurodegeneration and glia activation in the olfactory system of NPC1(-/-, which is accompanied by sensory deficits.

Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

DEFF Research Database (Denmark)

Maccarana, M.; Kalamajski, S.; Kongsgaard, M.

2009-01-01

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks...... of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican......-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found...

Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

Science.gov (United States)

Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Tawada, Akira; Suzuki, Osamu

2015-10-01

Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. © 2015 Wiley Periodicals, Inc.

Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins.

Science.gov (United States)

Mizumoto, Shuji; Yamada, Shuhei; Sugahara, Kazuyuki

2015-10-01

Recent functional studies on chondroitin sulfate-dermatan sulfate (CS-DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS-DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS-DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS-DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS-DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. Copyright © 2015. Published by Elsevier Ltd.

Are Niemann-Pick type C proteins key players in cnidarian-dinoflagellate endosymbioses?

Science.gov (United States)

Dani, Vincent; Ganot, Philippe; Priouzeau, Fabrice; Furla, Paola; Sabourault, Cecile

2014-09-01

The symbiotic interaction between cnidarians, such as corals and sea anemones, and the unicellular algae Symbiodinium is regulated by yet poorly understood cellular mechanisms, despite the ecological importance of coral reefs. These mechanisms, including host-symbiont recognition and metabolic exchange, control symbiosis stability under normal conditions, but also lead to symbiosis breakdown (bleaching) during stress. This study describes the repertoire of the sterol-trafficking proteins Niemann-Pick type C (NPC1 and NPC2) in the symbiotic sea anemone Anemonia viridis. We found one NPC1 gene in contrast to the two genes (NPC1 and NPC1L1) present in vertebrate genomes. While only one NPC2 gene is present in many metazoans, this gene has been duplicated in cnidarians, and we detected four NPC2 genes in A. viridis. However, only one gene (AvNPC2-d) was upregulated in symbiotic relative to aposymbiotic sea anemones and displayed higher expression in the gastrodermis (symbiont-containing tissue) than in the epidermis. We performed immunolabelling experiments on tentacle cross sections and demonstrated that the AvNPC2-d protein was closely associated with symbiosomes. In addition, AvNPC1 and AvNPC2-d gene expression was strongly downregulated during stress. These data suggest that AvNPC2-d is involved in both the stability and dysfunction of cnidarian-dinoflagellate symbioses. © 2014 John Wiley & Sons Ltd.

Niemann-pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis.

Science.gov (United States)

Millard, E E; Srivastava, K; Traub, L M; Schaffer, J E; Ory, D S

2000-12-08

The Niemann-Pick type C1 (NPC1) protein is a key participant in intracellular trafficking of low density lipoprotein cholesterol, but its role in regulation of sterol homeostasis is not well understood. To characterize further the function of NPC1, we generated stable Chinese hamster ovary (CHO) cell lines overexpressing the human NPC1 protein (CHO/NPC1). NPC1 overexpression increases the rate of trafficking of low density lipoprotein cholesterol to the endoplasmic reticulum and the rate of delivery of endosomal cholesterol to the plasma membrane (PM). CHO/NPC1 cells exhibit a 1.5-fold increase in total cellular cholesterol and up to a 2.9-fold increase in PM cholesterol. This increase in PM cholesterol is closely paralleled by a 3-fold increase in de novo cholesterol synthesis. Inhibition of cholesterol synthesis results in marked redistribution of PM cholesterol to intracellular sites, suggesting an unsuspected role for NPC1 in internalization of PM cholesterol. Despite elevated total cellular cholesterol, CHO/NPC1 cells exhibit increased cholesterol synthesis, which may be attributable to both resistance to oxysterol suppression of sterol-regulated gene expression and to reduced endoplasmic reticulum cholesterol levels under basal conditions. Taken together, these studies provide important new insights into the role of NPC1 in the determination of the levels and distribution of cellular cholesterol.

Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis

International Nuclear Information System (INIS)

Bame, K.J.; Kiser, C.S.; Esko, J.D.

1987-01-01

The authors have isolated Chinese hamster ovary cell mutants defective in proteoglycan synthesis by radiographic screening for cells unable to incorporate 35 SO 4 into acid-precipitable material. Some mutants did not incorporate 35 SO 4 into acid-precipitable material, whereas others incorporated about 3-fold less radioactivity. HPLC anion exchange chromatographic analysis of radiolabelled glycosaminoglycans isolated from these mutants revealed many are defective in heparan sulfate biosynthesis. Mutants 803 and 677 do not synthesize heparan sulfate, although they produce chondroitin sulfate: strain 803 makes chondroitin sulfate normally, whereas 677 overaccumulates chondroitin sulfate by a factor of three. These mutants fall into the same complementation group, suggesting that the mutations are allelic. A second group of heparan sulfate biosynthetic mutants, consisting of cell lines 625, 668 and 679, produce undersulfated heparan sulfate and normal chondroitin sulfate. Treatment of the chains with nitrous acid should determine the position of the sulfate groups along the chain. These mutants may define a complementation group that is defective in the enzymes which modify the heparan sulfate chain. To increase the authors repertoire of heparan sulfate mutants, they are presently developing an in situ enzyme assay to screen colonies replica plated on filter discs for sulfotransferase defects

Variations of pH as an additional tool in the analysis of crowded NMR spectra of fucosylated chondroitin sulfates.

Science.gov (United States)

Ustyuzhanina, Nadezhda E; Dmitrenok, Andrey S; Bilan, Maria I; Shashkov, Alexander S; Gerbst, Alexey G; Usov, Anatolii I; Nifantiev, Nikolay E

2016-03-24

The influence of pH variation on chemical shift values in NMR spectra of fucosylated chondroitin sulfates was studied using polysaccharides isolated from three sea cucumber species Apostichopus japonicus, Actinopyga mauritiana and Cucumaria japonica. The signals of glucuronic acid residues were found to be the most sensitive to pH changes in comparison to the chemical shifts of the sulfated galactosamine and fucosyl units, most of which were altered insignificantly. It was shown that in the presence of imidazole-HCl buffer (pH 7.2) NMR spectra of the polysaccharides from A. japonicus and A. mauritiana were sufficiently resolved, whereas under acidic conditions their (1)H NMR spectra were complicated by overlapping of H-1 signals of GlcA and GalNAc. In the case of polysaccharide from C. japonica bearing 3-O-fucosylated and 3-O-sulfated glucuronic acid residues in the backbone, acidification of the medium led to separation of H-1 signals of GlcA3S and GalNAc. Therefore, the combination of data obtained at different pH values may be useful for interpretation of overcrowded spectra of fucosylated chondroitin sulfates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate E.

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Panisadee Avirutnan

2007-11-01

Full Text Available Dengue virus (DENV nonstructural protein-1 (NS1 is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection.

Russian experience with injectable chondroitin sulfate and glucosamine sulfate: a review of clinical trials

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A. E. Karateev

2018-01-01

Full Text Available The widespread use of parenteral chondroprotectors is a feature of Russian medical practice. There are many drugs of this series in a Russian physician's arsenal, including chondroitin sulfate (CS,  glucosamine sulfate (GS, glycosaminoglycan-peptide complex, and  bioactive concentrate from small sea fish for intramuscular  injections. The paper analyzes Russian trials of the efficacy and  safety of two injectable formulations of CS and GS (ICS and IGS.  ICS was tested in 17 articles containing a total of 1639 patients with  osteoarthritis (OA, non-specific back pain (NBP, or shoulder  fractures and pain after stroke. Standard therapy (NSAIDs +  physiotherapy served as a control in the majority of the paper. In  these trials, the reductions in visual analog scale (VAS and WOMAC pain in OA treated with ICS averaged 58.2±22.3% and those were 26.1±14.7% in the control groups; the reductions in VAS NBP  reached an average of 87.1±16.8 and 62.2±21.7%, respectively.  ICS also showed a good effect in shoulder fractures and pain after a  stroke. The number of local adverse reactions after injections was  insignificant (4.4%; they did not threaten the health of patients and they caused ICS to be discontinued only in 3 cases. IGS was  investigated in two trials (n=154, which confirmed its efficacy (total pain relief >50% and relative safety. Thus, the data of Russian trials suggest that ICS and IGS have good therapeutic potential and favorable tolerance.

Basement membrane proteoglycans in glomerular morphogenesis: chondroitin sulfate proteoglycan is temporally and spatially restricted during development

DEFF Research Database (Denmark)

McCarthy, K J; Bynum, K; St John, P L

1993-01-01

We previously reported the presence of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG) in basement membranes of almost all adult tissues. However, an exception to this ubiquitous distribution was found in the kidney, where BM-CSPG was absent from the glomerular capillary......, the present study used light and electron microscopic immunohistochemistry to examine the distribution of BM-CSPG and basement membrane heparan sulfate proteoglycan (BM-HSPG) during prenatal and postnatal renal development in the rat. Our results show that the temporal and spatial pattern of expression of BM...

Chondroitin Sulfate-Rich Extract of Skate Cartilage Attenuates Lipopolysaccharide-Induced Liver Damage in Mice.

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Song, Yeong Ok; Kim, Mijeong; Woo, Minji; Baek, Jang-Mi; Kang, Keon-Hee; Kim, Sang-Ho; Roh, Seong-Soo; Park, Chan Hum; Jeong, Kap-Seop; Noh, Jeong-Sook

2017-06-15

The protective effects of a chondroitin sulfate-rich extract (CSE) from skate cartilage against lipopolysaccharide (LPS)-induced hepatic damage were investigated, and its mechanism of action was compared with that of chondroitin sulfate (CS) from shark cartilage. ICR mice were orally administrated 200 mg/kg body weight (BW) of CS or 400 mg/kg BW of CSE for 3 consecutive days, followed by a one-time intraperitoneal injection of LPS (20 mg/kg BW). The experimental groups were vehicle treatment without LPS injection (NC group), vehicle treatment with LPS injection (LPS group), CS pretreatment with LPS injection (CS group), and CSE pretreatment with LPS injection (CSE group). Hepatic antioxidant enzyme expression levels in the CS and CSE groups were increased relative to those in the LPS group. In LPS-insulted hepatic tissue, inflammatory factors were augmented relative to those in the NC group, but were significantly suppressed by pretreatment with CS or CSE. Moreover, CS and CSE alleviated the LPS-induced apoptotic factors and mitogen-activated protein kinase (MAPK). In addition, CS and CSE effectively decreased the serum lipid concentrations and downregulated hepatic sterol regulatory element-binding proteins expression. In conclusion, the skate CSE could protect against LPS-induced hepatic dyslipidemia, oxidative stress, inflammation, and apoptosis, probably through the regulation of MAPK signaling.

SRPX2 is a novel chondroitin sulfate proteoglycan that is overexpressed in gastrointestinal cancer.

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Kaoru Tanaka

Full Text Available SRPX2 (Sushi repeat-containing protein, X-linked 2 has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG. Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.

A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

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Han, Wenjun; Wang, Wenshuang; Zhao, Mei; Sugahara, Kazuyuki; Li, Fuchuan

2014-10-03

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Eliminase from a Marine Bacterium That Degrades Hyaluronan and Chondroitin Sulfate*

Science.gov (United States)

Han, Wenjun; Wang, Wenshuang; Zhao, Mei; Sugahara, Kazuyuki; Li, Fuchuan

2014-01-01

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ4,5HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications. PMID:25122756

Antithrombotic activities of fucosylated chondroitin sulfates and their depolymerized fragments from two sea cucumbers.

Science.gov (United States)

Liu, Xiaoxiao; Hao, Jiejie; Shan, Xindi; Zhang, Xiao; Zhao, Xiaoliang; Li, Qinying; Wang, Xiaojiang; Cai, Chao; Li, Guoyun; Yu, Guangli

2016-11-05

Fucosylated chondroitin sulfate (FCS), a glycosaminoglycan extracted from the body wall of sea cucumber, is a promising antithrombotic agent. The chemical structures of FCSc isolated from sea cucumber Cucumaria frondosa and its depolymerized fragment (dFCSc) were characterized for the first time. Additionally, anticoagulant and antithrombotic activities were evaluated in vitro and in vivo. The results demonstrated that dFCSc exhibited better antithrombotic-hemorrhagic ratio than native FCSc on the electrical induced arterial thrombosis model in rats. Compared to FCSt obtained from Thelenota ananas, FCSc possessed different sulfation patterns but similar antithrombotic effects. Therefore, sulfation pattern of FCS might not affect anticoagulation and antithrombosis as much as molecular weight may. Our results proposed a new point of view to understand the structure-activity relationship of FCS as alternative agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rapid acquisition of isolate-specific antibodies to chondroitin sulfate A-adherent Plasmodium falciparum isolates in Ghanaian primigravidae

DEFF Research Database (Denmark)

Cox, Sharon E; Staalsoe, Trine; Arthur, Paul

2005-01-01

RBC to chondroitin sulfate A (CSA). The VSA mediating CSA binding (VSA(CSA)) and thus sequestration of pRBC in the placenta are antigenically distinct from those that mediate pRBC sequestration elsewhere in the body, and it has been suggested that VSA(CSA) are relatively conserved and may thus constitute...

Spectral-fluorescent study of the interaction of the polymethine dye probe Cyan 2 with chondroitin-4-sulfate

Science.gov (United States)

Tatikolov, Alexander S.; Akimkin, Timofey M.; Panova, Ina G.; Yarmoluk, Sergiy M.

2017-04-01

The noncovalent interaction of the polymethine dye probe 3,3‧,9-trimethylthiacarbocyanine iodide (Cyan 2) with chondroitin-4-sulfate (C4S) in buffer solutions with different pH and in water in the absence of buffers has been studied by spectral-fluorescent methods. It has been shown that in all media studied, at relatively high concentrations, the dye is bound to C4S mainly as a monomer, which is accompanied by a steep rise of fluorescence (the intermediate formation of dye aggregates on the biopolymer is also observed). From the dependence of the fluorescence quantum yield on the concentration of C4S, the parameters of binding of the dye monomer to C4S were obtained: the effective binding constant K, the number of the monomeric C4S units n per one dye monomer bound to C4S, and the fluorescence quantum yield of the bound dye monomer Φfb. The dependence of Φfb (and K) on pH of the medium is not monotonic: it has a minimum in the region of neutral pH and a growth in the regions of acid and basic pH. This can be explained by changing the charge of a C4S macromolecule as a function of pH and related conformational alterations in the biopolymer, which can affect the rigidity of a dye molecule and the energy of its interaction with the biopolymer.

LC-MS n Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

Science.gov (United States)

Huang, Rongrong; Pomin, Vitor H.; Sharp, Joshua S.

2011-09-01

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS n . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS n fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS n experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS n methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.

The NTS-DBL2X region of VAR2CSA Induces cross-reactive antibodies that inhibit adhesion of several Plasmodium falciparum isolates to chondroitin sulfate A

DEFF Research Database (Denmark)

Bigey, Pascal; Gnidehou, Sédami; Doritchamou, Justin

2011-01-01

Background. Binding to chondroitin sulfate A by VAR2CSA, a parasite protein expressed on infected erythrocytes, allows placental sequestration of Plasmodium falciparum-infected erythrocytes. This leads to severe consequences such as maternal anemia, stillbirths, and intrauterine growth retardation....... The latter has been clearly associated to increased morbidity and mortality of the infants. Acquired anti-VAR2CSA antibodies have been associated with improved pregnancy outcomes, suggesting a vaccine could prevent the syndrome. However, identifying functionally important regions in the large VAR2CSA protein...

Association between Human Plasma Chondroitin Sulfate Isomers and Carotid Atherosclerotic Plaques

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Elisabetta Zinellu

2012-01-01

Full Text Available Several studies have evidenced variations in plasma glycosaminoglycans content in physiological and pathological conditions. In normal human plasma GAGs are present mainly as undersulfated chondroitin sulfate (CS. The aim of the present study was to evaluate possible correlations between plasma CS level/structure and the presence/typology of carotid atherosclerotic lesion. Plasma CS was purified from 46 control subjects and 47 patients undergoing carotid endarterectomy showing either a soft or a hard plaque. The concentration and structural characteristics of plasma CS were assessed by capillary electrophoresis of constituent unsaturated fluorophore-labeled disaccharides. Results showed that the concentration of total CS isomers was increased by 21.4% (P<0.01 in plasma of patients, due to a significant increase of undersulfated CS. Consequently, in patients the plasma CS charge density was significantly reduced with respect to that of controls. After sorting for plaque typology, we found that patients with soft plaques and those with hard ones differently contribute to the observed changes. In plasma from patients with soft plaques, the increase in CS content was not associated with modifications of its sulfation pattern. On the contrary, the presence of hard plaques was associated with CS sulfation pattern modifications in presence of quite normal total CS isomers levels. These results suggest that the plasma CS content and structure could be related to the presence and the typology of atherosclerotic plaque and could provide a useful diagnostic tool, as well as information on the molecular mechanisms responsible for plaque instability.

Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

International Nuclear Information System (INIS)

Duval, Caroline; Touche, Veronique; Tailleux, Anne; Fruchart, Jean-Charles; Fievet, Catherine; Clavey, Veronique; Staels, Bart; Lestavel, Sophie

2006-01-01

Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRα and LXRβ) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARα and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARα ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis

Facile synthesis of chondroitin sulfate-stabilized gold nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Li Wei, E-mail: weilixj8510@163.com [School of Science, Henan Agricultural University, No. 95 Wenhua Road, Zhengzhou, Henan 450002 (China); Li Xin; Su Hui; Zhao Shiju; Li Yanyun; Hu Jiandong [School of Science, Henan Agricultural University, No. 95 Wenhua Road, Zhengzhou, Henan 450002 (China)

2011-02-15

A facile and simple method for the synthesis of biocompatible gold nanoparticles (AuNPs) at room temperature has been developed by using sodium borohydride as the reducing agent and employing an inexpensive water-soluble chondroitin sulfate (CS) biopolymer as the stabilizing agent. The as-prepared AuNPs were characterized with ultraviolet-visible (UV-vis) spectroscopy and transmission electron microscopy (TEM). Additionally, the stability of AuNPs in aqueous solution was investigated as a function of the electrolyte sodium chloride concentration. The experimental results showed that even high sodium chloride concentration (1 M) also did not destabilize the colloidal gold solution. So it could be speculated that the high stability of AuNPs should be attributed to the electrostatic repulsion and steric hindrance between the AuNPs stabilized by CS molecules, which wrapped around the surface of as-prepared AuNPs and prevented their agglomeration, and simultaneously improve biocompatibility of AuNPs as well.

Facile synthesis of chondroitin sulfate-stabilized gold nanoparticles

International Nuclear Information System (INIS)

Li Wei; Li Xin; Su Hui; Zhao Shiju; Li Yanyun; Hu Jiandong

2011-01-01

A facile and simple method for the synthesis of biocompatible gold nanoparticles (AuNPs) at room temperature has been developed by using sodium borohydride as the reducing agent and employing an inexpensive water-soluble chondroitin sulfate (CS) biopolymer as the stabilizing agent. The as-prepared AuNPs were characterized with ultraviolet-visible (UV-vis) spectroscopy and transmission electron microscopy (TEM). Additionally, the stability of AuNPs in aqueous solution was investigated as a function of the electrolyte sodium chloride concentration. The experimental results showed that even high sodium chloride concentration (1 M) also did not destabilize the colloidal gold solution. So it could be speculated that the high stability of AuNPs should be attributed to the electrostatic repulsion and steric hindrance between the AuNPs stabilized by CS molecules, which wrapped around the surface of as-prepared AuNPs and prevented their agglomeration, and simultaneously improve biocompatibility of AuNPs as well.

In Vivo Anti-Cancer Mechanism of Low-Molecular-Weight Fucosylated Chondroitin Sulfate (LFCS) from Sea Cucumber Cucumaria frondosa.

Science.gov (United States)

Liu, Xiaoxiao; Liu, Yong; Hao, Jiejie; Zhao, Xiaoliang; Lang, Yinzhi; Fan, Fei; Cai, Chao; Li, Guoyun; Zhang, Lijuan; Yu, Guangli

2016-05-12

The low-molecular-weight fucosylated chondroitin sulfate (LFCS) was prepared from native fucosylated chondroitin sulfate (FCS), which was extracted and isolated from sea cucumber Cucumaria frondosa, and the anti-cancer mechanism of LFCS on mouse Lewis lung carcinoma (LLC) was investigated. The results showed that LFCS remarkably inhibited LLC growth and metastasis in a dose-dependent manner. LFCS induced cell cycle arrest by increasing p53/p21 expression and apoptosis through activation of caspase-3 activity in LLC cells. Meanwhile, LFCS suppressed the expression of vascular endothelial growth factor (VEGF), increased the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and downregulated the matrix metalloproteinases (MMPs) level. Furthermore, LFCS significantly suppressed the activation of ERK1/2/p38 MAPK/NF-κB pathway, which played a prime role in expression of MMPs. All of these data indicate LFCS may be used as anti-cancer drug candidates and deserve further study.

In Vivo Anti-Cancer Mechanism of Low-Molecular-Weight Fucosylated Chondroitin Sulfate (LFCS from Sea Cucumber Cucumaria frondosa

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Xiaoxiao Liu

2016-05-01

Full Text Available The low-molecular-weight fucosylated chondroitin sulfate (LFCS was prepared from native fucosylated chondroitin sulfate (FCS, which was extracted and isolated from sea cucumber Cucumaria frondosa, and the anti-cancer mechanism of LFCS on mouse Lewis lung carcinoma (LLC was investigated. The results showed that LFCS remarkably inhibited LLC growth and metastasis in a dose-dependent manner. LFCS induced cell cycle arrest by increasing p53/p21 expression and apoptosis through activation of caspase-3 activity in LLC cells. Meanwhile, LFCS suppressed the expression of vascular endothelial growth factor (VEGF, increased the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1 and downregulated the matrix metalloproteinases (MMPs level. Furthermore, LFCS significantly suppressed the activation of ERK1/2/p38 MAPK/NF-κB pathway, which played a prime role in expression of MMPs. All of these data indicate LFCS may be used as anti-cancer drug candidates and deserve further study.

Silk fibroin-chondroitin sulfate scaffold with immuno-inhibition property for articular cartilage repair.

Science.gov (United States)

Zhou, Feifei; Zhang, Xianzhu; Cai, Dandan; Li, Jun; Mu, Qin; Zhang, Wei; Zhu, Shouan; Jiang, Yangzi; Shen, Weiliang; Zhang, Shufang; Ouyang, Hong Wei

2017-11-01

The demand of favorable scaffolds has increased for the emerging cartilage tissue engineering. Chondroitin sulfate (CS) and silk fibroin have been investigated and reported with safety and excellent biocompatibility as tissue engineering scaffolds. However, the rapid degradation rate of pure CS scaffolds presents a challenge to effectively recreate neo-tissue similar to natural articular cartilage. Meanwhile the silk fibroin is well used as a structural constituent material because its remarkable mechanical properties, long-lasting in vivo stability and hypoimmunity. The application of composite silk fibroin and CS scaffolds for joint cartilage repair has not been well studied. Here we report that the combination of silk fibroin and CS could synergistically promote articular cartilage defect repair. The silk fibroin (silk) and silk fibroin/CS (silk-CS) scaffolds were fabricated with salt-leaching, freeze-drying and crosslinking methodologies. The biocompatibility of the scaffolds was investigated in vitro by cell adhesion, proliferation and migration with human articular chondrocytes. We found that silk-CS scaffold maintained better chondrocyte phenotype than silk scaffold; moreover, the silk-CS scaffolds reduced chondrocyte inflammatory response that was induced by interleukin (IL)-1β, which is in consistent with the well-documented anti-inflammatory activities of CS. The in vivo cartilage repair was evaluated with a rabbit osteochondral defect model. Silk-CS scaffold induced more neo-tissue formation and better structural restoration than silk scaffold after 6 and 12weeks of implantation in ICRS histological evaluations. In conclusion, we have developed a silk fibroin/ chondroitin sulfate scaffold for cartilage tissue engineering that exhibits immuno-inhibition property and can improve the self-repair capacity of cartilage. Severe cartilage defect such as osteoarthritis (OA) is difficult to self-repair because of its avascular, aneural and alymphatic nature

A dual-wavelength overlapping resonance Rayleigh scattering method for the determination of chondroitin sulfate with nile blue sulfate

Science.gov (United States)

Cui, Zhiping; Hu, Xiaoli; Liu, Shaopu; Liu, Zhongfang

2011-12-01

A dual-wavelength overlapping resonance Rayleigh scattering (DWO-RRS) method was developed to detect chondroitin sulfate (CS) with nile blue sulfate (NBS). At pH 3.0-4.0 Britton-Robinson (BR) buffer medium, CS interacted with NBS to form an ion-association complex. As a result, the new spectra of resonance Rayleigh scattering (RRS), second order scattering (SOS) and frequence doubling scattering (FDS) appeared and their intensities were enhanced greatly. Their maximum wavelengths were located at 303 nm (RRS), 362 nm (RRS), 588 nm (SOS) and 350 nm (FDS), respectively. The scattering intensities of the three methods were proportional to the concentration of CS in certain ranges. The methods had high sensitivity and the detection limits were between 1.5 and 7.1 ng mL -1. The DWO-RRS method had the highest sensitivity with the detection limit being 1.5 ng mL -1. The characteristics of the spectra and optimal reaction conditions of RRS method were investigated. The effects of coexistent substances on the determination of CS were evaluated. Owing to the high sensitivity, RRS method had been applied to the determination of CS in eye drops with satisfactory results. The recovery range was between 99.4% and 104.6% and the relative standard deviation (RSD) was between 0.4% and 0.8%. In addition, the reasons for RRS enhancement were discussed and the shape of ion-association complex was characterized by atomic force microscopy (AFM).

Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease.

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André R A Marques

Full Text Available Impaired function of NPC1 or NPC2 lysosomal proteins leads to the intracellular accumulation of unesterified cholesterol, the primary defect underlying Niemann-Pick type C (NPC disease. In addition, glycosphingolipids (GSLs accumulate in lysosomes as well. Intralysosomal lipid accumulation triggers the activation of a set of genes, including potential biomarkers. Transcript levels of Gpnmb have been shown to be elevated in various tissues of an NPC mouse model. We speculated that Gpnmb could serve as a marker for visceral lipid accumulation in NPC disease. We report that Gpnmb expression is increased at protein level in macrophages in the viscera of Npc1nih/nih mice. Interestingly, soluble Gpnmb was also found to be increased in murine and NPC patient plasma. Exposure of RAW264.7 macrophages to the NPC-phenotype-inducing drug U18666A also upregulated Gpnmb expression. Inhibition of GSL synthesis with the glucosylceramide synthase (GCS inhibitor N-butyl-1-deoxynojirimycin prevented U18666A-induced Gpnmb induction and secretion. In summary, we show that Gpnmb is upregulated in NPC mice and patients, most likely due to GSL accumulation.

A thermo-responsive and photo-polymerizable chondroitin sulfate-based hydrogel for 3D printing applications.

Science.gov (United States)

Abbadessa, A; Blokzijl, M M; Mouser, V H M; Marica, P; Malda, J; Hennink, W E; Vermonden, T

2016-09-20

The aim of this study was to design a hydrogel system based on methacrylated chondroitin sulfate (CSMA) and a thermo-sensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate)-polyethylene glycol triblock copolymer (M15P10) as a suitable material for additive manufacturing of scaffolds. CSMA was synthesized by reaction of chondroitin sulfate with glycidyl methacrylate (GMA) in dimethylsulfoxide at 50°C and its degree of methacrylation was tunable up to 48.5%, by changing reaction time and GMA feed. Unlike polymer solutions composed of CSMA alone (20% w/w), mixtures based on 2% w/w of CSMA and 18% of M15P10 showed strain-softening, thermo-sensitive and shear-thinning properties more pronounced than those found for polymer solutions based on M15P10 alone. Additionally, they displayed a yield stress of 19.2±7.0Pa. The 3D printing of this hydrogel resulted in the generation of constructs with tailorable porosity and good handling properties. Finally, embedded chondrogenic cells remained viable and proliferating over a culture period of 6days. The hydrogel described herein represents a promising biomaterial for cartilage 3D printing applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Niemann-Pick C1 (NPC1/NPC1-like1 Chimeras Define Sequences Critical for NPC1’s Function as a Filovirus Entry Receptor

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Esther Ndungo

2012-10-01

Full Text Available We recently demonstrated that Niemann-Pick C1 (NPC1, a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1, an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus receptor activity. We exploited the structural similarity between NPC1 and NPC1L1 to construct and analyze a panel of chimeras in which NPC1L1 sequences were replaced with cognate sequences from NPC1. Only one chimera, NPC1L1 containing the second luminal domain (C of NPC1 in place of its own, bound to the viral glycoprotein, GP. This engineered protein mediated authentic filovirus infection nearly as well as wild-type NPC1, and more efficiently than did a minimal NPC1 domain C-based receptor recently described by us. A reciprocal chimera, NPC1 containing NPC1L1’s domain C, was completely inactive. Remarkably, an intra-domain NPC1L1-NPC1 chimera bearing only a ~130-amino acid N–terminal region of NPC1 domain C could confer substantial viral receptor activity on NPC1L1. Taken together, these findings account for the failure of NPC1L1 to serve as a filovirus receptor, highlight the central role of the luminal domain C of NPC1 in filovirus entry, and reveal the direct involvement of N–terminal domain C sequences in NPC1’s function as a filovirus receptor.

A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate

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Restaino, Odile Francesca, E-mail: odilefrancesca.restaino@unina2.it [Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples (Italy); Finamore, Rosario, E-mail: rosario.finamore@unina2.it [Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples (Italy); Diana, Paola, E-mail: paola.diana@unina2.it [Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples (Italy); Marseglia, Mariacarmela, E-mail: marimars84@hotmail.it [Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples (Italy); Vitiello, Mario, E-mail: mariovitiello.ita@gmail.com [Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples (Italy); Casillo, Angela, E-mail: angela.casillo@unina.it [Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples (Italy); Bedini, Emiliano, E-mail: emiliano.bedini@unina.it [Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples (Italy); Parrilli, Michelangelo, E-mail: michelangelo.parrilli@unina.it [Department of Biology, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples (Italy); and others

2017-03-15

Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core. - Highlights: • A multi-analytical approach was set up, for the first time, for the determination of the residual keratan sulfate

A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate

International Nuclear Information System (INIS)

Restaino, Odile Francesca; Finamore, Rosario; Diana, Paola; Marseglia, Mariacarmela; Vitiello, Mario; Casillo, Angela; Bedini, Emiliano; Parrilli, Michelangelo

2017-01-01

Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core. - Highlights: • A multi-analytical approach was set up, for the first time, for the determination of the residual keratan sulfate

Radiolabeling procedure, quality control and stability of 99mTc-labeled chondroitin sulfate: A new approach of targeting osteoarthritis

International Nuclear Information System (INIS)

Sobal, Grazyna; Menzel, Ernst Johannes; Sinzinger, Helmut

2008-01-01

Chondroitin sulfate (CS) is an endogenous component of cartilage which could determine osteoarthritis after radiolabeling. Radiolabeling was performed by 99m TcO 4 - /tin method. We found that pH is a limiting factor. At pH 6.2 in 0.05 M Na acetate buffer 32.8% colloid was formed, at pH 5.0 in 0.5 M Na acetate, in our methodology only 4.7% colloid. The tracer was highly stable. We conclude that 99m TcCS could be a promising imaging agent of osteoarthritis due to simple radiolabeling and high stability

Synthesis of the oligosaccharides related to branching sites of fucosylated chondroitin sulfates from sea cucumbers.

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Ustyuzhanina, Nadezhda E; Fomitskaya, Polina A; Gerbst, Alexey G; Dmitrenok, Andrey S; Nifantiev, Nikolay E

2015-02-02

Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have started systematic synthesis of oligosaccharides with well-defined structure related to various fragments of these polysaccharides. In this communication, the synthesis of non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to branching sites of FCS is described. The target compounds are built up of propyl β-d-glucuronic acid residue bearing at O-3 α-l-fucosyl or α-l-fucosyl-(1→3)-α-l-fucosyl substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected according to the known to date holothurian FCS structures. Stereospecific α-glycoside bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-l-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the remote participation of the chloroacetyl groups with the formation of the stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the α-side. The experimental results were in good agreement with the SCF/MP2 calculated energies of such participation. The synthesized oligosaccharides are regarded as model compounds for the determination of a structure-activity relationship in FCS.

A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate.

Science.gov (United States)

Restaino, Odile Francesca; Finamore, Rosario; Diana, Paola; Marseglia, Mariacarmela; Vitiello, Mario; Casillo, Angela; Bedini, Emiliano; Parrilli, Michelangelo; Corsaro, Maria Michela; Trifuoggi, Marco; De Rosa, Mario; Schiraldi, Chiara

2017-03-15

Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder, Niemann-Pick type C1 disease.

Science.gov (United States)

Sarkar, Sovan; Maetzel, Dorothea; Korolchuk, Viktor I; Jaenisch, Rudolf

2014-06-01

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.

A pharmacoproteomic study confirms the synergistic effect of chondroitin sulfate and glucosamine.

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Calamia, Valentina; Mateos, Jesús; Fernández-Puente, Patricia; Lourido, Lucía; Rocha, Beatriz; Fernández-Costa, Carolina; Montell, Eulalia; Vergés, Josep; Ruiz-Romero, Cristina; Blanco, Francisco J

2014-06-10

Osteoarthritis (OA) is the most common age-related rheumatic disease. Chondrocytes play a primary role in mediating cartilage destruction and extracellular matrix (ECM) breakdown, which are main features of the OA joint. Quantitative proteomics technologies are demonstrating a very interesting power for studying the molecular effects of some drugs currently used to treat OA patients, such as chondroitin sulfate (CS) and glucosamine (GlcN). In this work, we employed the iTRAQ (isobaric tags for relative and absolute quantitation) technique to assess the effect of CS and GlcN, both alone and in combination, in modifying cartilage ECM metabolism by the analysis of OA chondrocytes secretome. 186 different proteins secreted by the treated OA chondrocytes were identified. 36 of them presented statistically significant differences (p ≤ 0.05) between untreated and treated samples: 32 were increased and 4 decreased. The synergistic chondroprotective effect of CS and GlcN, firstly reported by our group at the intracellular level, is now demonstrated also at the extracellular level.

Protective effect of exogenous chondroitin 4,6-sulfate in the acute degradation of articular cartilage in the rabbit.

Science.gov (United States)

Uebelhart, D; Thonar, E J; Zhang, J; Williams, J M

1998-05-01

The injection of 2.0 mg chymopapain into the adolescent rabbit knee causes severe loss of articular cartilage proteoglycans (PG). Although chondrocytes attempt to restore lost PG, failure to repair ensues. Pure chondroitin 4,6-sulfate (Condrosulf, IBSA Lugano, Switzerland) has been used in clinical studies of human osteoarthritis (OA) as a slow-acting drug for OA (SYSADOA). Using our model of articular cartilage injury, we examined the effects of oral and intramuscular administration of Condrosulf after chymopapain-induced cartilage injury. In this study, animals received an injection of 2.0 mg chymopapain (Chymodiactin, Boots Pharmaceuticals) into the left knee and were sacrificed after 84 days. The contralateral right knee served as a noninjected control. Some animals received oral Condrosulf while others received intramuscular injections of Condrosulf. Serum keratan sulfate (KS) levels were monitored to ensure degradation of the cartilage PG. Those animals not exhibiting at least a 100% increase of serum KS following chymopapain injection were excluded from the study. At sacrifice, cartilage PG contents were markedly reduced in animals receiving an injection of 2.0 mg chymopapain with no further treatment. In contrast, oral administration of Condrosulf beginning 11 days prior to chymopapain injury resulted in significantly higher (P = 0.0036) cartilage PG contents. Intramuscular administration of Condrosulf resulted in higher, but less significantly so (P = 0.0457), cartilage PG contents. These results suggest that daily Condrosulf treatment prior to and continuing after chymopapain injury may have a protective effect on the damaged cartilage, allowing it to continue to re-synthesize matrix PG after the treatment is discontinued.

Cholesterol pathways affected by small molecules that decrease sterol levels in Niemann-Pick type C mutant cells.

Directory of Open Access Journals (Sweden)

Madalina Rujoi

2010-09-01

Full Text Available Niemann-Pick type C (NPC disease is a genetically inherited multi-lipid storage disorder with impaired efflux of cholesterol from lysosomal storage organelles.The effect of screen-selected cholesterol lowering compounds on the major sterol pathways was studied in CT60 mutant CHO cells lacking NPC1 protein. Each of the selected chemicals decreases cholesterol in the lysosomal storage organelles of NPC1 mutant cells through one or more of the following mechanisms: increased cholesterol efflux from the cell, decreased uptake of low-density lipoproteins, and/or increased levels of cholesteryl esters. Several chemicals promote efflux of cholesterol to extracellular acceptors in both non-NPC and NPC1 mutant cells. The uptake of low-density lipoprotein-derived cholesterol is inhibited by some of the studied compounds.Results herein provide the information for prioritized further studies in identifying molecular targets of the chemicals. This approach proved successful in the identification of seven chemicals as novel inhibitors of lysosomal acid lipase (Rosenbaum et al, Biochim. Biophys. Acta. 2009, 1791:1155-1165.

In vivo evaluation of hybrid patches composed of PLA based copolymers and collagen/chondroitin sulfate for ligament tissue regeneration.

Science.gov (United States)

Pinese, Coline; Gagnieu, Christian; Nottelet, Benjamin; Rondot-Couzin, Capucine; Hunger, Sylvie; Coudane, Jean; Garric, Xavier

2017-10-01

Biomaterials for soft tissues regeneration should exhibit sufficient mechanical strength, demonstrating a mechanical behavior similar to natural tissues and should also promote tissues ingrowth. This study was aimed at developing new hybrid patches for ligament tissue regeneration by synergistic incorporation of a knitted structure of degradable polymer fibers to provide mechanical strength and of a biomimetic matrix to help injured tissues regeneration. PLA- Pluronic ® (PLA-P) and PLA-Tetronic ® (PLA-T) new copolymers were shaped as knitted patches and were associated with collagen I (Coll) and collagen I/chondroitine-sulfate (Coll CS) 3-dimensional matrices. In vitro study using ligamentocytes showed the beneficial effects of CS on ligamentocytes proliferation. Hybrid patches were then subcutaneously implanted in rats for 4 and 12 weeks. Despite degradation, patches retained strength to answer the mechanical physiological needs. Tissue integration capacity was assessed with histological studies. We showed that copolymers, associated with collagen and chondroitin sulfate sponge, exhibited very good tissue integration and allowed neotissue synthesis after 12 weeks in vivo. To conclude, PLA-P/CollCS and PLA-T/CollCS hybrid patches in terms of structure and composition give good hopes for tendon and ligament regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1778-1788, 2017. © 2016 Wiley Periodicals, Inc.

Spatiotemporal expression of chondroitin sulfate sulfotransferases in the postnatal developing mouse cerebellum.

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Ishii, Maki; Maeda, Nobuaki

2008-08-01

Chondroitin sulfate (CS) proteoglycans are major components of the cell surface and the extracellular matrix in the developing brain and bind to various proteins via CS chains in a CS structure-dependent manner. This study demonstrated the expression pattern of three CS sulfotransferase genes, dermatan 4-O-sulfotransferase (D4ST), uronyl 2-O-sulfotransferase (UST), and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), in the mouse postnatal cerebellum. These sulfotransferases are responsible for the biosynthesis of oversulfated structures in CS chains such as B, D, and E units, which constitute the binding sites for various heparin-binding proteins. Real-time reverse transcription-polymerase chain reaction analysis indicated that the expression of UST increased remarkably during cerebellar development. The amounts of B and D units, which are generated by UST activity, in the cerebellar CS chains also increased during development. In contrast, the expression of GalNAc4S-6ST and its biosynthetic product, E unit, decreased during postnatal development. In situ hybridization experiments revealed the levels of UST and GalNAc4S-6ST mRNAs to correlate inversely in many cells including Purkinje cells, granule cells in the external granular layer, and inhibitory interneurons. In these neurons, the expression of UST increased and that of GalNAc4S-6ST decreased during development and/or maturation. D4ST was also expressed by many neurons, but its expression was not simply correlated with development, which might contribute to the diversification of CS structures expressed by distinct neurons. These results suggest that the CS structures of various cerebellar neurons change during development and such changes of CS are involved in the regulation of various signaling pathways.

EGFR Activation Mediates Inhibition of Axon Regeneration by Myelin and Chondroitin Sulfate Proteoglycans

Science.gov (United States)

Koprivica, Vuk; Cho, Kin-Sang; Park, Jong Bae; Yiu, Glenn; Atwal, Jasvinder; Gore, Bryan; Kim, Jieun A.; Lin, Estelle; Tessier-Lavigne, Marc; Chen, Dong Feng; He, Zhigang

2005-10-01

Inhibitory molecules associated with myelin and the glial scar limit axon regeneration in the adult central nervous system (CNS), but the underlying signaling mechanisms of regeneration inhibition are not fully understood. Here, we show that suppressing the kinase function of the epidermal growth factor receptor (EGFR) blocks the activities of both myelin inhibitors and chondroitin sulfate proteoglycans in inhibiting neurite outgrowth. In addition, regeneration inhibitors trigger the phosphorylation of EGFR in a calcium-dependent manner. Local administration of EGFR inhibitors promotes significant regeneration of injured optic nerve fibers, pointing to a promising therapeutic avenue for enhancing axon regeneration after CNS injury.

Liver and Skin Histopathology in Adults with Acid Sphingomyelinase Deficiency (Niemann-Pick Disease Type B)

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Thurberg, Beth L.; Wasserstein, Melissa P.; Schiano, Thomas; O’Brien, Fanny; Richards, Susan; Cox, Gerald F.; McGovern, Margaret M.

2012-01-01

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder characterized by the pathologic accumulation of sphingomyelin in multiple cells types, and occurs most prominently within the liver, spleen and lungs, leading to significant clinical disease. Seventeen ASMD patients underwent a liver biopsy during baseline screening for a Phase 1 trial of recombinant human acid sphingomyelinase (rhASM) in adults with Niemann-Pick disease type B. Eleven of the 17 were enrolled in the trial and each received a single dose of rhASM and underwent a repeat liver biopsy on Day 14. Biopsies were evaluated for fibrosis, sphingomyelin accumulation and macrophage infiltration by light and electron microscopy. When present, fibrosis was periportal and pericellular, predominantly surrounding affected Kupffer cells. Two baseline biopsies exhibited frank cirrhosis. Sphingomyelin was localized to isolated Kupffer cells in mildly affected biopsies and was present in both Kupffer cells and hepatocytes in more severely affected cases. Morphometric quantification of sphingomyelin storage in liver biopsies ranged from 4–44% of the microscopic field. Skin biopsies were also performed at baseline and Day 14 in order to compare the sphingomyelin distribution in a peripheral tissue to that of liver. Sphingomyelin storage was present at lower levels in multiple cell types of the skin, including dermal fibroblasts, macrophages, vascular endothelial cells, vascular smooth muscle cells and Schwann cells. This Phase 1 trial of rhASM in adults with ASMD provided a unique opportunity for a prospective assessment of hepatic and skin pathology in this rare disease and their potential usage as pharmacodynamic biomarkers. PMID:22613999

Radiolabeling procedure, quality control and stability of {sup 99m}Tc-labeled chondroitin sulfate: A new approach of targeting osteoarthritis

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Sobal, Grazyna [Department of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna, Waehringer Guertel 18-20 (Austria)], E-mail: Grazyna.Sobal@meduniwien.ac.at; Menzel, Ernst Johannes [Institute of Immunology, Medical University of Vienna, Vienna (Austria); Sinzinger, Helmut [Department of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna, Waehringer Guertel 18-20 (Austria)

2008-04-15

Chondroitin sulfate (CS) is an endogenous component of cartilage which could determine osteoarthritis after radiolabeling. Radiolabeling was performed by {sup 99m}TcO{sub 4}{sup -}/tin method. We found that pH is a limiting factor. At pH 6.2 in 0.05 M Na acetate buffer 32.8% colloid was formed, at pH 5.0 in 0.5 M Na acetate, in our methodology only 4.7% colloid. The tracer was highly stable. We conclude that {sup 99m}TcCS could be a promising imaging agent of osteoarthritis due to simple radiolabeling and high stability.

Chondroitin Sulfate Is Indispensable for Pluripotency and Differentiation of Mouse Embryonic Stem Cells

Science.gov (United States)

Izumikawa, Tomomi; Sato, Ban; Kitagawa, Hiroshi

2014-01-01

Chondroitin sulfate (CS) proteoglycans are present on the surfaces of virtually all cells and in the extracellular matrix and are required for cytokinesis at early developmental stages. Studies have shown that heparan sulfate (HS) is essential for maintaining mouse embryonic stem cells (ESCs) that are primed for differentiation, whereas the function of CS has not yet been elucidated. To clarify the role of CS, we generated glucuronyltransferase-I-knockout ESCs lacking CS. We found that CS was required to maintain the pluripotency of ESCs and promoted initial ESC commitment to differentiation compared with HS. In addition, CS-A and CS-E polysaccharides, but not CS-C polysaccharides, bound to E-cadherin and enhanced ESC differentiation. Multiple-lineage differentiation was inhibited in chondroitinase ABC-digested wild-type ESCs. Collectively, these results suggest that CS is a novel determinant in controlling the functional integrity of ESCs via binding to E-cadherin.

Case Report: Ursodeoxycholic acid treatment in Niemann-Pick disease type C; clinical experience in four cases

Science.gov (United States)

Movsesyan, Nina; Platt, Frances M.

2017-01-01

In this case series, we demonstrate that Ursodeoxycholic acid (UDCA) improves liver dysfunction in Niemann-Pick type C (NPC) and may restore a suppressed cytochrome p450 system. NPC disease is a progressive neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 genes. Liver disease is a common feature presenting either acutely as cholestatic jaundice in the neonatal period, or in later life as elevated liver enzymes indicative of liver dysfunction. Recently, an imbalance in bile acid synthesis in a mouse model of NPC disease was linked to suppression of the P450 detoxification system and was corrected by UDCA treatment. UDCA (3α, 7β-dihydroxy-5β-cholanic acid), a hydrophilic bile acid, is used to treat various cholestatic disorders. In this report we summarise the findings from four independent cases of NPC, three with abnormal liver enzyme levels at baseline, that were subsequently treated with UDCA. The patients differed in age and clinical features, they all tolerated the drug well, and in those with abnormal liver function, there were significant improvements in their liver enzyme parameters. PMID:29119141

Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection.

Science.gov (United States)

Côté, Marceline; Misasi, John; Ren, Tao; Bruchez, Anna; Lee, Kyungae; Filone, Claire Marie; Hensley, Lisa; Li, Qi; Ory, Daniel; Chandran, Kartik; Cunningham, James

2011-08-24

Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann-Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.

Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate

International Nuclear Information System (INIS)

Huang, Zhao; Nooeaid, Patcharakamon; Kohl, Benjamin; Roether, Judith A.; Schubert, Dirk W.; Meier, Carola; Boccaccini, Aldo R.; Godkin, Owen; Ertel, Wolfgang; Arens, Stephan; Schulze-Tanzil, Gundula

2015-01-01

To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1–2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~ 93–95% with a mean pore sizes of 237 ± 48 μm (Alg) and 197 ± 61 μm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell–cell contacts. - Highlights: • Alginate foam scaffolds revealed a high porosity and mean pore size of 197–237 μm. • Chondroitin sulfate was released over 14 days by the scaffolds. • Chondrocytes

Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate

Energy Technology Data Exchange (ETDEWEB)

Huang, Zhao [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Nooeaid, Patcharakamon [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Kohl, Benjamin [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Roether, Judith A.; Schubert, Dirk W. [Institute of Polymer Materials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Meier, Carola [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Boccaccini, Aldo R. [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Godkin, Owen; Ertel, Wolfgang; Arens, Stephan [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Schulze-Tanzil, Gundula, E-mail: gundula.schulze@pmu.ac.at [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Institute of Anatomy, Paracelsus Medical University, Nuremberg (Germany)

2015-05-01

To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1–2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~ 93–95% with a mean pore sizes of 237 ± 48 μm (Alg) and 197 ± 61 μm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell–cell contacts. - Highlights: • Alginate foam scaffolds revealed a high porosity and mean pore size of 197–237 μm. • Chondroitin sulfate was released over 14 days by the scaffolds. • Chondrocytes

Chondroitin sulfate-functionalized polyamidoamine as a tumor-targeted carrier for miR-34a delivery.

Science.gov (United States)

Chen, Wenqi; Liu, Yong; Liang, Xiao; Huang, Yu; Li, Quanshun

2017-07-15

Chondroitin sulfate (CS) was modified on a polyamidoamine dendrimer (PAMAM) through Michael addition to construct a tumor-targeted carrier CS-PAMAM for miR-34a delivery. The derivative CS-PAMAM was demonstrated to achieve an efficient cellular uptake of miR-34a in a CD44-dependent endocytosis way and further facilitate the endosomal escape of miR-34a after 4h. Through the miR-34a delivery, obvious inhibition of cell proliferation could be detected which was attributed to the enhancement of cell apoptosis and cell cycle arrest, and meanwhile the cell migration and invasion has been observed to be inhibited. Finally, the intravenous injection of CS-PAMAM/miR-34a formulation into mice bearing human lung adenocarcinoma cell A549 xenografts could efficiently inhibit the tumor growth and induce the tumor apoptosis owing to the enhanced accumulation of miR-34a in tumor tissue. Overall, CS-PAMAM is potential to be used as a tumor-targeted oligonucleotide carrier for achieving tumor gene therapy. The cationic dendrimer PAMAM was modified by chondroitin sulfate (CS) through Michael addition to construct a tumor-targeted carrier CS-PAMAM for miR-34a delivery. The introduction of CS could achieve an efficient cellular uptake and intracellular transfection of miR-34a in a CD44-dependent endocytosis manner. The miR-34a delivery could execute the anti-proliferation activity by simultaneously inducing cell apoptosis and cell cycle arrest, and also the anti-migration activity. The CS-PAMAM-mediated systemic delivery of miR-34a showed significant inhibition of tumor growth and induction of tumor apoptosis using a mice model of subcutaneously implanted tumors. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Effect of chondroitin sulfate on osteogenetic differentiation of human mesenchymal stem cells

International Nuclear Information System (INIS)

Schneiders, Wolfgang; Rentsch, Claudia; Rehberg, Sebastian; Rein, Susanne; Zwipp, Hans; Rammelt, Stefan

2012-01-01

Chondroitin sulfate (CS) has anti-inflammatory properties and increases the regeneration ability of injured bone. In different in vivo investigations on bone defects the addition of CS to calcium phosphate bone cement has lead to an enhanced bone remodeling and increased new bone formation. The goal of this study was to evaluate the cellular effects of CS on human mesenchymal stem cells (hMSCs). In cell culture experiments hMSCs were incubated on calcium phosphate bone cements with and without CS and cultivated in a proliferation and an osteogenetic differentiation media. Alkaline phosphatase and the proliferation rate were determined on days 1, 7 and 14. Concerning the proliferation rates, no significant differences were detected. On days 1, 7 and 14 a significantly higher activity of alkaline phosphatase, an early marker of osteogenesis, was detected around CS modified cements in both types of media. The addition of CS leads to a significant increase of osteogenetic differentiation of hMSCs. To evaluate the influence of the osteoconductive potency of CS in twelve adult male Wistar rats, the interface reaction of cancellous bone to a nanocrystalline hydroxyapatite cement containing type I collagen (CDHA/Coll) without and with CS (CDHA/Coll/CS) was evaluated. Cylindrical implants were inserted press-fit into a defect of the tibial head. 28 days after the operation the direct bone contact and the percentage of newly formed bone were significantly higher on CDHA/Coll/CS-implants (p < 0.05). The addition of CS appears to enhance new bone formation on CDHA/Coll-composites in the early stages of bone healing. Possible mechanisms are discussed. - Highlights: ► The influence of chondroitin sulfate (CS) on bone metabolism was evaluated. ► CS leads to a significant increase of osteogenetic differentiation of hMSCs. ► In small animal investigation CS seems to enhance osteogenesis in bone healing.

Prophylactic vesical instillations with 0.2% chondroitin sulfate may reduce symptoms of acute radiation cystitis in patients undergoing radiotherapy for gynecological malignancies

NARCIS (Netherlands)

Hazewinkel, M.H.; Stalpers, L.J.A.; Dijkgraaf, M.G.; Roovers, J.P.W.R.

2011-01-01

We studied the feasibility and efficacy of intravesical instillations with 40 ml chondroitin sulfate 0.2% solution to prevent or reduce acute radiation cystitis in women undergoing pelvic radiotherapy. In a comparative pilot study in 20 patients, half of the patients received instillations.

Homologous overexpression of RfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide.

Science.gov (United States)

Cimini, Donatella; De Rosa, Mario; Carlino, Elisabetta; Ruggiero, Alessandro; Schiraldi, Chiara

2013-05-09

Glycosaminoglycans, such as hyaluronic acid, heparin, and chondroitin sulfate, are among the top ranked products in industrial biotechnology for biomedical applications, with a growing world market of billion dollars per year. Recently a remarkable progress has been made in the development of tailor-made strains as sources for the manufacturing of such products. The genetic modification of E. coli K4, a natural producer of chondroitin sulfate precursor, is challenging considering the lack of detailed information on its genome, as well as its mobilome. Chondroitin sulfate is currently used as nutraceutical for the treatment of osteoarthritis, and several new therapeutic applications, spanning from the development of skin substitutes to live attenuated vaccines, are under evaluation. E. coli K4 was used as host for the overexpression of RfaH, a positive regulator that controls expression of the polysaccharide biosynthesis genes and other genes necessary for the virulence of E. coli K4. Various engineering strategies were compared to investigate different types of expression systems (plasmid vs integrative cassettes) and integration sites (genome vs endogenous mobile element). All strains analysed in shake flasks on different media showed a capsular polysaccharide production improved by 40 to 140%, compared to the wild type, with respect to the final product titer. A DO-stat fed-batch process on the 2L scale was also developed for the best performing integrative strain, EcK4r3, yielding 5.3 g ∙ L(-1) of K4 polysaccharide. The effect of rfaH overexpression in EcK4r3 affected the production of lipopolysaccharide and the expression of genes involved in the polysaccharide biosynthesis pathway (kfoC and kfoA), as expected. An alteration of cellular metabolism was revealed by changes of intracellular pools of UDP-sugars which are used as precursors for polysaccharide biosynthesis. The present study describes the identification of a gene target and the application of a

Fucosylated chondroitin sulfate oligosaccharides exert anticoagulant activity by targeting at intrinsic tenase complex with low FXII activation: Importance of sulfation pattern and molecular size.

Science.gov (United States)

Li, Junhui; Li, Shan; Yan, Lufeng; Ding, Tian; Linhardt, Robert J; Yu, Yanlei; Liu, Xinyue; Liu, Donghong; Ye, Xingqian; Chen, Shiguo

2017-10-20

Fucosylated chondroitin sulfates (fCSs) are structurally unusual glycosaminoglycans isolated from sea cucumbers that exhibit potent anticoagulant activity. These fCSs were isolated from sea cucumber, Isostichopus badionotus and Pearsonothuria graeffei. Fenton reaction followed by gel filtration chromatography afforded fCS oligosaccharides, with different sulfation patterns identified by mass and NMR spectroscopy, and these were used to clarify the relationship between the structures and the anticoagulant activities of fCSs. In vitro activities were measured by activated partial thromboplastin time (APTT), thrombin time (TT), thrombin and factor Xa inhibition, and activation of FXII. The results showed that free radicals preferentially acted on GlcA residues affording oligosaccharides that were purified from both fCSs. The inhibition of thrombin and factor X activities, mediated through antithrombin III and heparin cofactor II of fCSs oligosaccharides were affected by their molecular weight and fucose branches. Oligosaccharides with different sulfation patterns of the fucose branching had a similar ability to inhibit the FXa by the intrinsic factor Xase (factor IXa-VIIIa complex). Oligosaccharides with 2,4-O-sulfo fucose branches from fCS-Ib showed higher activities than ones with 3,4-O-disulfo branches obtained from fCS-Pg. Furthermore, a heptasaccharide is the minimum size oligosaccharide required for anticoagulation and FXII activation. This activity was absent for fCS oligosaccharides smaller than nonasaccharides. Molecular size and fucose branch sulfation are important for anticoagulant activity and reduction of size can reverse the activation of FXII caused by native fCSs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

DEFF Research Database (Denmark)

Faassen, A E; Schrager, J A; Klein, D J

1992-01-01

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell...... collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG...... was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related...

Overproduction, purification and crystallization of a chondroitin sulfate A-binding DBL domain from a Plasmodium falciparum var2csa-encoded PfEMP1 protein

International Nuclear Information System (INIS)

Higgins, Matthew K.

2008-01-01

A chondroitin sulfate A-binding DBL important in placental malaria has been overproduced, purified and crystallized. Diffraction data were collected to 1.9 Å resolution. The PfEMP1 proteins of the malaria parasite Plasmodium falciparum are inserted into the membrane of infected red blood cells, where they mediate adhesion to a variety of human receptors. The DBL domains of the var2csa-encoded PfEMP1 protein play a critical role in malaria of pregnancy, tethering infected cells to the surface of the placenta through interactions with the glycosaminoglycan carbohydrate chondroitin sulfate A (CSA). A CSA-binding DBL domain has been overproduced in a bacterial expression system, purified and crystallized. Native data sets extending to 1.9 Å resolution have been collected and phasing is under way

Overproduction, purification and crystallization of a chondroitin sulfate A-binding DBL domain from a Plasmodium falciparum var2csa-encoded PfEMP1 protein

Energy Technology Data Exchange (ETDEWEB)

Higgins, Matthew K., E-mail: mkh20@cam.ac.uk [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom)

2008-03-01

A chondroitin sulfate A-binding DBL important in placental malaria has been overproduced, purified and crystallized. Diffraction data were collected to 1.9 Å resolution. The PfEMP1 proteins of the malaria parasite Plasmodium falciparum are inserted into the membrane of infected red blood cells, where they mediate adhesion to a variety of human receptors. The DBL domains of the var2csa-encoded PfEMP1 protein play a critical role in malaria of pregnancy, tethering infected cells to the surface of the placenta through interactions with the glycosaminoglycan carbohydrate chondroitin sulfate A (CSA). A CSA-binding DBL domain has been overproduced in a bacterial expression system, purified and crystallized. Native data sets extending to 1.9 Å resolution have been collected and phasing is under way.

Annexins as organizers of cholesterol- and sphingomyelin-enriched membrane microdomains in Niemann-Pick type C disease.

Science.gov (United States)

Domon, Magdalena; Nasir, Mehmet Nail; Matar, Gladys; Pikula, Slawomir; Besson, Françoise; Bandorowicz-Pikula, Joanna

2012-06-01

Growing evidence suggests that membrane microdomains enriched in cholesterol and sphingomyelin are sites for numerous cellular processes, including signaling, vesicular transport, interaction with pathogens, and viral infection, etc. Recently some members of the annexin family of conserved calcium and membrane-binding proteins have been recognized as cholesterol-interacting molecules and suggested to play a role in the formation, stabilization, and dynamics of membrane microdomains to affect membrane lateral organization and to attract other proteins and signaling molecules onto their territory. Furthermore, annexins were implicated in the interactions between cytosolic and membrane molecules, in the turnover and storage of cholesterol and in various signaling pathways. In this review, we focus on the mechanisms of interaction of annexins with lipid microdomains and the role of annexins in membrane microdomains dynamics including possible participation of the domain-associated forms of annexins in the etiology of human lysosomal storage disease called Niemann-Pick type C disease, related to the abnormal storage of cholesterol in the lysosome-like intracellular compartment. The involvement of annexins and cholesterol/sphingomyelin-enriched membrane microdomains in other pathologies including cardiac dysfunctions, neurodegenerative diseases, obesity, diabetes mellitus, and cancer is likely, but is not supported by substantial experimental observations, and therefore awaits further clarification.

The Effect of Glucosamine and/or Chondroitin Sulfate on the Progression of Knee Osteoarthritis: A GAIT Report

Science.gov (United States)

Sawitzke, Allen D; Shi, Helen; Finco, Martha; Dunlop, Dorothy D; Bingham, Clifton O; Harris, Crystal L; Singer, Nora G; Bradley, John D; Silver, David; Jackson, Christopher G; Lane, Nancy E; Oddis, Chester V; Wolfe, Fred; Lisse, Jeffrey; Furst, Daniel E; Reda, Domenic J; Moskowitz, Roland W; Williams, H James; Clegg, Daniel O

2010-01-01

Objective Osteoarthritis of the knee causes significant morbidity and current medical treatment is limited to symptom relief, as therapies able to slow structural damage remain elusive. This study sought to evaluate the effect of glucosamine hydrochloride (glucosamine, G), sodium chondroitin sulfate (chondroitin sulfate, CS) (alone and in combination), celecoxib and placebo on progressive loss of joint space width (JSW). Methods A double-blind twenty-four month placebo-controlled study conducted at nine sites in the United States enrolled 572 participants from Glucosamine/chondroitin Arthritis Intervention Trial (GAIT) who satisfied radiographic criteria (Kellgren and Lawrence (K&L) Grade 2 or 3 changes and JSW of at least 2mm at baseline). Persons with primarily lateral compartment narrowing at any time point were excluded. Patients continued G 500mg three times daily, CS 400mg three times daily, the combination, celecoxib 200mg daily or placebo as randomized for GAIT. Minimum medial tibiofemoral JSW was measured at baseline, 12 and 24 months. The primary outcome measure was JSW change from baseline. Results The average JSW loss at 2 years for placebo, adjusted for design and clinical factors, was 0.16mm. No statistically significant difference for any treatment group compared to the placebo group was observed. Treatment effects for K&L Grade 2 knees, but not K&L Grade 3 knees showed a trend toward improvement relative to placebo. The study’s power was diminished by sample size, variance of JSW measurement and a smaller than expected loss in JSW. Conclusions At two years, no treatment achieved a predefined clinically important difference in JSW loss compared to placebo. However, patients with K&L Grade 2 osteoarthritis appear to have the greatest potential for modification by these treatments (ClinicalTrials.gov number, NCT00032890). PMID:18821708

Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury

NARCIS (Netherlands)

Bartus, Katalin; James, Nicholas D; Didangelos, Athanasios; Bosch, Karen D; Verhaagen, J.; Yáñez-Muñoz, Rafael J; Rogers, John H; Schneider, Bernard L; Muir, Elizabeth M; Bradbury, Elizabeth J

2014-01-01

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate

Age-related changes in the incorporation of [35S]sulfate into two proteoglycan populations from human cartilage

International Nuclear Information System (INIS)

Triphaus, G.F.; Schmidt, A.; Buddecke, E.

1980-01-01

From human hyaline cartilage (processus xyphoid) preincubated in the presence of [ 35 S] sulfate, proteoglycans were extracted by 4M guanidinium chloride and divided into 6 age groups. Fractionation of proteoglycans by gel filtration under dissociative conditions resulted in two proteoglycan fractions (a and b) with different hydrodynamic volumes. The higher molecular weight fraction a contained chondroitin sulfate, the fraction b keratan sulfate as predominant glycosaminoglycan, the chondroitin sulfate/keratan sulfate ratio decreasing with increasing age in either fraction. The relative portion of proteoglycan fraction b and its 35 S-labelling increased with increasing age. From the specific 35 S radioactivities of the chondroitin sulfate and keratan sulfate preparations, the occurrence of two independent proteoglycan populations is suggested. A precursorproduct relationship between proteoglycan fraction a and b could be excluded. (orig.)

Effect of chondroitin sulfate on osteogenetic differentiation of human mesenchymal stem cells

Energy Technology Data Exchange (ETDEWEB)

Schneiders, Wolfgang, E-mail: schneidersw@gmx.de; Rentsch, Claudia; Rehberg, Sebastian; Rein, Susanne; Zwipp, Hans; Rammelt, Stefan

2012-10-01

Chondroitin sulfate (CS) has anti-inflammatory properties and increases the regeneration ability of injured bone. In different in vivo investigations on bone defects the addition of CS to calcium phosphate bone cement has lead to an enhanced bone remodeling and increased new bone formation. The goal of this study was to evaluate the cellular effects of CS on human mesenchymal stem cells (hMSCs). In cell culture experiments hMSCs were incubated on calcium phosphate bone cements with and without CS and cultivated in a proliferation and an osteogenetic differentiation media. Alkaline phosphatase and the proliferation rate were determined on days 1, 7 and 14. Concerning the proliferation rates, no significant differences were detected. On days 1, 7 and 14 a significantly higher activity of alkaline phosphatase, an early marker of osteogenesis, was detected around CS modified cements in both types of media. The addition of CS leads to a significant increase of osteogenetic differentiation of hMSCs. To evaluate the influence of the osteoconductive potency of CS in twelve adult male Wistar rats, the interface reaction of cancellous bone to a nanocrystalline hydroxyapatite cement containing type I collagen (CDHA/Coll) without and with CS (CDHA/Coll/CS) was evaluated. Cylindrical implants were inserted press-fit into a defect of the tibial head. 28 days after the operation the direct bone contact and the percentage of newly formed bone were significantly higher on CDHA/Coll/CS-implants (p < 0.05). The addition of CS appears to enhance new bone formation on CDHA/Coll-composites in the early stages of bone healing. Possible mechanisms are discussed. - Highlights: Black-Right-Pointing-Pointer The influence of chondroitin sulfate (CS) on bone metabolism was evaluated. Black-Right-Pointing-Pointer CS leads to a significant increase of osteogenetic differentiation of hMSCs. Black-Right-Pointing-Pointer In small animal investigation CS seems to enhance osteogenesis in bone healing.

Placental sequestration of Plasmodium falciparum malaria parasites is mediated by the interaction between VAR2CSA and chondroitin sulfate A on syndecan-1

DEFF Research Database (Denmark)

Ayres Pereira, Marina; Mandel Clausen, Thomas; Pehrson, Caroline

2016-01-01

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans......-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor...... for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial...

Genetic dissection of a cell-autonomous neurodegenerative disorder: lessons learned from mouse models of Niemann-Pick disease type C

Directory of Open Access Journals (Sweden)

Manuel E. Lopez

2013-09-01

Full Text Available Understanding neurodegenerative disease progression and its treatment requires the systematic characterization and manipulation of relevant cell types and molecular pathways. The neurodegenerative lysosomal storage disorder Niemann-Pick disease type C (NPC is highly amenable to genetic approaches that allow exploration of the disease biology at the organismal, cellular and molecular level. Although NPC is a rare disease, genetic analysis of the associated neuropathology promises to provide insight into the logic of disease neural circuitry, selective neuron vulnerability and neural-glial interactions. The ability to control the disorder cell-autonomously and in naturally occurring spontaneous animal models that recapitulate many aspects of the human disease allows for an unparalleled dissection of the disease neurobiology in vivo. Here, we review progress in mouse-model-based studies of NPC disease, specifically focusing on the subtype that is caused by a deficiency in NPC1, a sterol-binding late endosomal membrane protein involved in lipid trafficking. We also discuss recent findings and future directions in NPC disease research that are pertinent to understanding the cellular and molecular mechanisms underlying neurodegeneration in general.

Modelling the cancer growth process by Stochastic Differential Equations with the effect of Chondroitin Sulfate (CS) as anticancer therapeutics

Science.gov (United States)

Syahidatul Ayuni Mazlan, Mazma; Rosli, Norhayati; Jauhari Arief Ichwan, Solachuddin; Suhaity Azmi, Nina

2017-09-01

A stochastic model is introduced to describe the growth of cancer affected by anti-cancer therapeutics of Chondroitin Sulfate (CS). The parameters values of the stochastic model are estimated via maximum likelihood function. The numerical method of Euler-Maruyama will be employed to solve the model numerically. The efficiency of the stochastic model is measured by comparing the simulated result with the experimental data.

Chondroitin sulfate and glucosamine in the cartilage and subchondral bone repair of dogs - Histological findings

Directory of Open Access Journals (Sweden)

R.B. Eleotério

2015-04-01

Full Text Available Chondroitin and glucosamine sulfate nutraceuticals are commonly used in the management of degenerative articular disease in veterinary routine. However, there are controversies on the contribution of these substances to articular cartilage. The purpose of this study was to evaluate the efficiency of a chondroitin and glucosamine sulfate-based veterinary nutraceutical on the repair of an induced osteochondral defect in a dog femoral condyle, by macroscopic, histological and histomorphometric analyses. The nutraceutical was orally administered the day following injury induction, every 24 hours (treated group, TG, n=24, compared with animals that did not receive the product (control group, CG, n=24. Six animals per group were anaesthetized for sample collection at 15, 30, 60 and 90 days after surgery. At 15 days, defects were macroscopically filled with red-pinkish tissue. After 30 days, whitish color tissue was observed, both in TG and CG animals, with firmer consistency to touch at 60 and 90 postoperative days. Histological analysis demonstrated that, in both groups, there was initial blood clot formation, which was subsequently substituted by a fibrin net, with capillary proliferation from the adjacent bone marrow and infiltration of mesenchymal cells in clot periphery. As cellular differentiation developed, repair tissue presented a fibrocartilage aspect most of the time, and new subchondral bone formation occurred in the deepest area corresponding to the defect. Histomorphometry suggested that the nutraceutical did not favor the articular cartilage repair process. It was concluded that nutraceutical did not significantly influence chondrocytes proliferation or hyaline architecture restoration.

Chondroitin sulfate immobilization at the surface of electrospun nanofiber meshes for cartilage tissue regeneration approaches

International Nuclear Information System (INIS)

Piai, Juliana Francis; Alves da Silva, Marta; Martins, Albino; Torres, Ana Bela; Faria, Susana

2017-01-01

Highlights: • Chemical immobilization of chondroitin sulfate at the surface of nanofiber meshes. • CS-immobilized NFMs showed lower roughness and higher hydrophilicity. • CS-immobilized NFMs offer a highly effective substrate for hACs phenotypic stability. - Abstract: Aiming at improving the biocompatibility of biomaterial scaffolds, surface modification presents a way to preserve their mechanical properties and to improve the surface bioactivity. In this work, chondroitin sulfate (CS) was immobilized at the surface of electrospun poly(caprolactone) nanofiber meshes (PCL NFMs), previously functionalized by UV/O_3 exposure and aminolysis. Contact angle, SEM, optical profilometry, FTIR, X-ray photoelectron spectroscopy techniques confirmed the success of CS-immobilization in PCL NFMs. Furthermore, CS-immobilized PCL NFMs showed lower roughness and higher hydrophilicity than the samples without CS. Human articular chondrocytes (hACs) were cultured on electrospun PCL NFMs with or without CS immobilization. It was observed that hACs proliferated through the entire time course of the experiment in both types of nanofibrous scaffolds, as well as for the production of glycosaminoglycans. Quantitative-PCR results demonstrated over-expression of cartilage-related genes such as Aggrecan, Collagen type II, COMP and Sox9 on both types of nanofibrous scaffolds. Morphological observations from SEM and LSCM revealed that hACs maintained their characteristic round shape and cellular agglomeration exclusively on PCL NFMs with CS immobilization. In conclusion, CS immobilization at the surface of PCL NFMs was achieved successfully and provides a valid platform enabling further surface functionalization methods in scaffolds to be developed for cartilage tissue engineering.

Chondroitin sulfate immobilization at the surface of electrospun nanofiber meshes for cartilage tissue regeneration approaches

Energy Technology Data Exchange (ETDEWEB)

Piai, Juliana Francis [3B’s Research Group − Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães (Portugal); ICVS/3B’s − PT Government Associate Laboratory, Braga/Guimarães (Portugal); Grupo de Materiais Poliméricos e Compósitos, GMPC – Departamento de Química- Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900, Maringá, Paraná (Brazil); Alves da Silva, Marta; Martins, Albino; Torres, Ana Bela [3B’s Research Group − Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães (Portugal); ICVS/3B’s − PT Government Associate Laboratory, Braga/Guimarães (Portugal); Faria, Susana [Research Center Officinal Mathematical, Department of Mathematics for Science and Technology, University of Minho, Campus de Azurém, 4800-058 Guimarães (Portugal); and others

2017-05-01

Highlights: • Chemical immobilization of chondroitin sulfate at the surface of nanofiber meshes. • CS-immobilized NFMs showed lower roughness and higher hydrophilicity. • CS-immobilized NFMs offer a highly effective substrate for hACs phenotypic stability. - Abstract: Aiming at improving the biocompatibility of biomaterial scaffolds, surface modification presents a way to preserve their mechanical properties and to improve the surface bioactivity. In this work, chondroitin sulfate (CS) was immobilized at the surface of electrospun poly(caprolactone) nanofiber meshes (PCL NFMs), previously functionalized by UV/O{sub 3} exposure and aminolysis. Contact angle, SEM, optical profilometry, FTIR, X-ray photoelectron spectroscopy techniques confirmed the success of CS-immobilization in PCL NFMs. Furthermore, CS-immobilized PCL NFMs showed lower roughness and higher hydrophilicity than the samples without CS. Human articular chondrocytes (hACs) were cultured on electrospun PCL NFMs with or without CS immobilization. It was observed that hACs proliferated through the entire time course of the experiment in both types of nanofibrous scaffolds, as well as for the production of glycosaminoglycans. Quantitative-PCR results demonstrated over-expression of cartilage-related genes such as Aggrecan, Collagen type II, COMP and Sox9 on both types of nanofibrous scaffolds. Morphological observations from SEM and LSCM revealed that hACs maintained their characteristic round shape and cellular agglomeration exclusively on PCL NFMs with CS immobilization. In conclusion, CS immobilization at the surface of PCL NFMs was achieved successfully and provides a valid platform enabling further surface functionalization methods in scaffolds to be developed for cartilage tissue engineering.

Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe.

Science.gov (United States)

Mehta, Pramod Kumar; Lee, Hyeri; Lee, Keun-Hyeung

2017-05-15

The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R 2 =0.99) in aqueous solutions, 2.98nM (R 2 =0.98) in 1% serum samples, and 3.43nM (R 2 =0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response. Copyright © 2017 Elsevier B.V. All rights reserved.

Prognostic significance of highly sulfated chondroitin sulfates in ovarian cancer defined by the single chain antibody GD3A11.

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van der Steen, Sophieke C H A; van Tilborg, Angela A G; Vallen, Myrtille J E; Bulten, Johan; van Kuppevelt, Toin H; Massuger, Leon F A G

2016-03-01

The extracellular matrix (ECM) of ovarian cancer may provide a number of potential biomarkers. Chondroitin sulfate (CS), a class of sulfated polysaccharides, is abundantly present in the ECM of ovarian cancer. Structural alterations of CS chains (i.e. sulfation pattern) have been demonstrated to play a role in cancer development and progression. In this study we investigate the potential of highly sulfated CS as a biomarker in ovarian cancer using the single chain antibody GD3A11 selected by the phage display technology. The specificity of the antibody was determined by an indirect ELISA. GD3A11 epitope expression was assessed by immunohistochemistry in healthy organs, benign and malignant ovarian tumors (N=359) and correlated to clinical parameters. The CHST15 gene, responsible for the biosynthesis of highly sulfated CS was evaluated for mutation and methylation status. The GD3A11 epitope was minimally expressed in normal organs. Intense expression was observed in the ECM of different ovarian cancer subtypes, in contrast to benign ovarian tumors. Expression was independent of tumor grade, FIGO stage, and the use chemotherapy. For the aggressive ovarian cancer phenotype, intense expression was identified as an independent predictor for poor prognosis. CHST15 gene analysis showed no mutations nor an altered methylation status. Specific highly sulfated CS motifs expressed in the tumoral ECM hold biomarker potential in ovarian cancer patients. These matrix motifs constitute a novel class of biomarkers with prognostic significance and may be instrumental for innovative diagnostic and therapeutic applications (e.g. targeted therapy) in management of ovarian cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Age-related changes in the incorporation of (/sup 35/S)sulfate into two proteoglycan populations from human cartilage

Energy Technology Data Exchange (ETDEWEB)

Triphaus, G.F.; Schmidt, A.; Buddecke, E.

1980-12-01

From human hyaline cartilage (processus xyphoid) preincubated in the presence of (/sup 35/S) sulfate, proteoglycans were extracted by 4M guanidinium chloride and divided into 6 age groups. Fractionation of proteoglycans by gel filtration under dissociative conditions resulted in two proteoglycan fractions (a and b) with different hydrodynamic volumes. The higher molecular weight fraction a contained chondroitin sulfate, the fraction b keratan sulfate as predominant glycosaminoglycan, the chondroitin sulfate/keratan sulfate ratio decreasing with increasing age in either fraction. The relative portion of proteoglycan fraction b and its /sup 35/S-labelling increased with increasing age. From the specific /sup 35/S radioactivities of the chondroitin sulfate and keratan sulfate preparations, the occurrence of two independent proteoglycan populations is suggested. A precursorproduct relationship between proteoglycan fraction a and b could be excluded.

Changes in Caries Risk and Activity of a 9-Year-Old Patient with Niemann-Pick Disease Type C

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Késsia Suênia Fidelis Mesquita-Guimarães

2015-01-01

Full Text Available Objective. This case report describes the changes in caries risk and activity and dental treatment of a 9-year-old patient who presented with signs and symptoms of Niemann-Pick disease type C (NPC. Treatment. The preventive dental treatment included instructions to caregivers for oral hygiene and diet. A calcium hydroxide pulpotomy and restorative dental treatments were performed in a dental office with desensitization techniques and behavioral management. The patient was attended every 3 months for the control of dental plaque biofilm, for topical fluoride application, and for observing the pulpotomized tooth. Results. The bacterial plaque biofilm was being adequately controlled by the caregiver. After 2 years, the clinical and radiographic examination of the pulpotomized tooth showed the absence of internal root resorption and bone rarefaction, and clinical examination showed tooth sensitivity, dental pain, and gingival swelling. Conclusion. The pulpotomy prevented clinical and radiographic success. Dentists must be aware of and be able to identify systemic and local aspects associated with caries risk of children with NPC disease. Furthermore, dentists must employ stringent preventive measures and provide instructions to caregivers to reduce caries risk.

Reduction of VLDL secretion decreases cholesterol excretion in niemann-pick C1-like 1 hepatic transgenic mice.

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Stephanie M Marshall

Full Text Available An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL support TICE, antisense oligonucleotides (ASO were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP, which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg mice, which predominantly excrete cholesterol via TICE, and wild type (WT littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.

Chitosan-hyaluronan/nano chondroitin sulfate ternary composite sponges for medical use.

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Anisha, B S; Sankar, Deepthi; Mohandas, Annapoorna; Chennazhi, K P; Nair, Shantikumar V; Jayakumar, R

2013-02-15

In this work chitosan-hyaluronan composite sponge incorporated with chondroitin sulfate nanoparticle (nCS) was developed. The fabrication of hydrogel was based on simple ionic cross-linking using EDC, followed by lyophilization to obtain the composite sponge. nCS suspension was characterized using DLS and SEM and showed a size range of 100-150 nm. The composite sponges were characterized using SEM, FT-IR and TG-DTA. Porosity, swelling, biodegradation, blood clotting and platelet activation of the prepared sponges were also evaluated. Nanocomposites showed a porosity of 67% and showed enhanced swelling and blood clotting ability. Cytocompatibility and cell adhesion studies of the sponges were done using human dermal fibroblast (HDF) cells and the nanocomposite sponges showed more than 90% viability. Nanocomposite sponges also showed enhanced proliferation of HDF cells within two days of study. These results indicated that this nanocomposite sponges would be a potential candidate for wound dressing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Layer-by-layer assembly of type I collagen and chondroitin sulfate on aminolyzed PU for potential cartilage tissue engineering application

International Nuclear Information System (INIS)

He Xianyun; Wang Yingjun; Wu Gang

2012-01-01

Highlights: ► A novel biodegradable polyurethane (PU) was successfully synthesized. ► Surface aminolyzing of the PU was performed by reacting it with 1,3-propanediamine. ► Collagen and chondroitin sulfate were deposited alternately on the PU surface. - Abstract: In this paper, a two-step method was used to synthesize a biodegradable polyurethane (PU) composed of L-lysine ethyl ester diisocyanate (LDI), poly(ε-caprolactone) diols (PCL-diol) and 1,4:3,6-dianhydro-D-sorbitol (isosorbide). Amino groups were introduced onto the surface of the PU membrane by an amination reacting with 1,3-propanediamine to produce polycationic substratum. And then, type I collagen (Col) and chondroitin sulfate (CS) were deposited alternately on the polycationic substratum through layer-by-layer (LBL) assembly technology. The FTIR and 1 H NMR results showed that the polyurethane was successfully synthesized. Rhodamine B isothiocyanate (RBITC) fluorescence spectrum indicated that amino groups were successfully introduced onto the PU surface. The results of quartz-crystal microbalance (QCM) and RBITC-Col fluorescence spectroscopy monitoring the LBL assemble process presented that the Col/CS deposited alternately on the PU surface. X-ray photoelectron spectroscopy (XPS) results displayed that the CS deposited on the PU surface as well. The surface of the assembled PU became even smoother observed from the surface morphology by atomic force microscopy (AFM) imaging. The hydrophilicity of the PU membrane was greatly enhanced though the modification of LBL assembly. The PU modified with the adsorption of Col/CS may be a potential application for cartilage tissue engineering due to its created mimicking chondrogenic environment.

Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans.

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Noborn, Fredrik; Gomez Toledo, Alejandro; Green, Anders; Nasir, Waqas; Sihlbom, Carina; Nilsson, Jonas; Larson, Göran

2016-10-03

Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans.

Chondroitin sulfate-derivatized agarose beads: a new system for studying cation binding to glycosaminoglycans

International Nuclear Information System (INIS)

Hunter, G.K.

1987-01-01

Chondroitin sulfate (CS) has been covalently attached to aminoethyl-agarose beads in a carbodiimide-catalyzed reaction. In this process, an amide bond is formed between carboxylate groups on the glycosaminoglycan (GAG) and the primary amine groups of the beads. Under optimal conditions, up to 160 micrograms of CS is attached per milligram of beads. CS-agarose beads have been used to study Ca binding to GAGs. The beads are mixed with a solution containing CaCl 2 and 45 Ca and allowed to sediment under unit gravity. An aliquot of supernatant is then removed and 45 Ca activity is determined to quantitate remaining (free) Ca. Using this system, it was shown that CS binds approximately 0.7 Ca/disaccharide unit at saturation. Under the conditions used, the apparent association constant (KA) is approximately 14 mM. In principle, this derivatization protocol may be used to attach any proteoglycan or GAG (except keratan sulfate) to an insoluble support. CS-agarose beads provide a rapid, simple, and relatively artifact-free system for studying cation-GAG interactions

Deglycosylation of chondroitin sulfate proteoglycan and derived peptides

International Nuclear Information System (INIS)

Campbell, S.C.; Krueger, R.C.; Schwartz, N.B.

1990-01-01

In order to define the domain structure of proteoglycans as well as identify primary amino acid sequences specific for attachment of the various carbohydrate substituents, reliable techniques for deglycosylating proteoglycans are required. In this study, deglycosylation of cartilage chondroitin sulfate proteoglycan (CSPG) with minimal core protein cleavage was accomplished by digestion with chondroitinase ABC and keratanase, followed by treatment with anhydrous HF in pyridine. Nearly complete deglycosylation of secreted proteoglycan was verified within 45 min of HF treatment by loss of incorporated [ 3 H]glucosamine label from the proteoglycan as a function of time of treatment, as well as by direct analysis of carbohydrate content and xylosyltransferase acceptor activity of unlabeled core protein preparations. The deglycosylated CSPG preparations were homogeneous and of high molecular weight. Comparison of the intact deglycosylated core protein preparations with newly synthesized unprocessed precursors suggested that extensive proteolytic cleavage of the core protein did not occur during normal intracellular processing. Furthermore, peptide patterns generated after clostripain digestion of core protein precursor and of deglycosylated secreted proteoglycan were comparable. With the use of the clostripain digestion procedure, peptides were produced from unlabeled proteoglycan, and two predominant peptides from the most highly glycosylated regions were isolated, characterized, and deglycosylated. These peptides were found to follow similar kinetics of deglycosylation and to acquire xylose activity comparable to the intact core protein

Ataxia, dystonia and myoclonus in adult patients with Niemann-Pick type C.

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Koens, L H; Kuiper, A; Coenen, M A; Elting, J W J; de Vries, J J; Engelen, M; Koelman, J H T M; van Spronsen, F J; Spikman, J M; de Koning, T J; Tijssen, M A J

2016-09-01

Niemann-Pick type C (NP-C) is a rare autosomal recessive progressive neurodegenerative disorder caused by mutations in the NP-C 1 or 2 gene. Besides visceral symptoms, presentation in adolescent and adult onset variants is often with neurological symptoms. The most frequently reported presenting symptoms of NP-C in adulthood are psychiatric symptoms (38 %), cognitive decline (23 %) and ataxia (20 %). Myoclonus can be present, but its value in early diagnosis and the evolving clinical phenotype in NP-C is unclear. In this paper we present eight Dutch cases of NP-C of whom five with myoclonus. Eight patients with genetically confirmed NP-C were recruited from two Dutch University Medical Centers. A structured interview and neuropsychological tests (for working and verbal memory, attention and emotion recognition) were performed. Movement disorders were assessed using a standardized video protocol. Quality of life was evaluated by questionnaires (Rand-36, SIP-68, HAQ). In four of the five patients with myoclonic jerks simultaneous EEG with EMG was performed. A movement disorder was the initial neurological symptom in six patients: three with myoclonus and three with ataxia. Two others presented with psychosis. Four experienced cognitive deficits early in the course of the disease. Patients showed cognitive deficits in all investigated domains. Five patients showed myoclonic jerks, including negative myoclonus. In all registered patients EEG-EMG coherence analysis and/or back-averaging proved a cortical origin of myoclonus. Patients with more severe movement disorders experienced significantly more physical disabilities. Presenting neurological symptoms of NP-C include movement disorders, psychosis and cognitive deficits. At current neurological examination movement disorders were seen in all patients. The incidence of myoclonus in our cohort was considerably higher (63 %) than in previous publications and it was the presenting symptom in 38 %. A cortical origin

Is intravesical instillation of hyaluronic acid and chondroitin sulfate useful in preventing recurrent bacterial cystitis? A multicenter case control analysis

OpenAIRE

Giorgio Gugliotta; Gloria Calagna; Giorgio Adile; Salvatore Polito; Salvatore Saitta; Patrizia Speciale; Stefano Palomba; Antonino Perino; Roberta Granese; Biagio Adile

2015-01-01

Objective: Urinary tract infections (UTIs) are common in the female population and, over a lifetime, about half of women have at least one episode of UTI requiring antibiotic therapy. The aim of the current study was to compare two different strategies for preventing recurrent bacterial cystitis: intravesical instillation of hyaluronic acid (HA) plus chondroitin sulfate (CS), and antibiotic prophylaxis with sulfamethoxazole plus trimethoprim. Materials and methods: This was a retrospective...

Characterization and anti-tumor effects of chondroitin sulfate-chitosan nanoparticles delivery system

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Hu, Chieh-Shen; Tang, Sung-Ling; Chiang, Chiao-Hsi; Hosseinkhani, Hossein; Hong, Po-Da; Yeh, Ming-Kung

2014-11-01

We prepared chondroitin sulfate (ChS)-chitosan (CS) nanoparticles (NPs) as a delivery carrier, and doxorubicin (Dox) was used as a model drug. The physicochemical properties and biological activities of the Dox-ChS-CS NPs including the release profile, cell cytotoxicity, cellular internalization, and in vivo anti-tumor effects were evaluated. The ChS-CS NPs and Dox-ChS-CS NPs had a mean size of 262.0 ± 15.0 and 369.4 ± 77.4 nm, and a zeta potential of 30.2 ± 0.9 and 20.6 ± 3.1 mV, respectively. In vitro release tests showed that the 50 % release time for the Dox-ChS-CS NPs was 20 h. Two hepatoma cell models, HepG2 and HuH6, were used for evaluating the cytotoxicity and cell uptake efficiency of the Dox-ChS-CS NPs. A significant difference was observed between doxorubicin solution and the Dox-ChS-CS NPs in the cellular uptake within 60 min ( p < 0.01). For the in vivo human xenograft-nude mouse model, the Dox-ChS-CS NPs were more effective with less body weight loss and anti-tumor growth suppression in comparison with the Dox solution. The prepared Dox-ChS-CS NPs offer a new effective targeting nanoparticle delivery system platform for anti-tumor therapy.

Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.

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Panagos, Charalampos G; Thomson, Derek S; Moss, Claire; Hughes, Adam D; Kelly, Maeve S; Liu, Yan; Chai, Wengang; Venkatasamy, Radhakrishnan; Spina, Domenico; Page, Clive P; Hogwood, John; Woods, Robert J; Mulloy, Barbara; Bavington, Charlie D; Uhrín, Dušan

2014-10-10

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcβ4,6S(1 → 4) [FucαX(1 → 3)]GlcAβ(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial GSH replenishment as a potential therapeutic approach for Niemann Pick type C disease

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Sandra Torres

2017-04-01

Full Text Available Niemann Pick type C (NPC disease is a progressive lysosomal storage disorder caused by mutations in genes encoding NPC1/NPC2 proteins, characterized by neurological defects, hepatosplenomegaly and premature death. While the primary biochemical feature of NPC disease is the intracellular accumulation of cholesterol and gangliosides, predominantly in endolysosomes, mitochondrial cholesterol accumulation has also been reported. As accumulation of cholesterol in mitochondria is known to impair the transport of GSH into mitochondria, resulting in mitochondrial GSH (mGSH depletion, we investigated the impact of mGSH recovery in NPC disease. We show that GSH ethyl ester (GSH-EE, but not N-acetylcysteine (NAC, restored the mGSH pool in liver and brain of Npc1-/- mice and in fibroblasts from NPC patients, while both GSH-EE and NAC increased total GSH levels. GSH-EE but not NAC increased the median survival and maximal life span of Npc1-/- mice. Moreover, intraperitoneal therapy with GSH-EE protected against oxidative stress and oxidant-induced cell death, restored calbindin levels in cerebellar Purkinje cells and reversed locomotor impairment in Npc1-/- mice. High-resolution respirometry analyses revealed that GSH-EE improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1-/- mice. Lipidomic analyses showed that GSH-EE treatment had not effect in the profile of most sphingolipids in liver and brain, except for some particular species in brain of Npc1-/- mice. These findings indicate that the specific replenishment of mGSH may be a potential promising therapy for NPC disease, worth exploring alone or in combination with other options.

Clinical efficacy and safety over two years use of glucosamine, chondroitin sulfate, their combination, celecoxib or placebo taken to treat osteoarthritis of the knee: a GAIT report

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Shi, Helen; Finco, Martha F; Dunlop, Dorothy D; Harris, Crystal L; Singer, Nora G; Bradley, John D; Silver, David; Jackson, Christopher G; Lane, Nancy E; Oddis, Chester V; Wolfe, Fred; Lisse, Jeffrey; Furst, Daniel E; Bingham, Clifton O; Reda, Domenic J; Moskowitz, Roland W; Williams, H James; Clegg, Daniel O

2011-01-01

Objectives Osteoarthritis (OA) of the knee is a major cause of pain and limited function in older adults. Longer-term studies of medical therapy of OA are uncommon. This study was undertaken to evaluate the efficacy and safety of glucosamine and chondroitin sulfate (CS), alone or in combination, as well as celecoxib and placebo on painful knee OA over 24 months. Methods A 24-month, double-blind, placebo controlled study, conducted at 9 sites in the United States ancillary to the Glucosamine/Chondroitin Arthritis Intervention Trial (GAIT), enrolled 662 patients with knee OA who satisfied radiographic criteria (Kellgren/ Lawrence [K/L] grade 2 or grade 3 changes and JSW of at least 2 mm at baseline). Patients who had been randomized to 1 of the 5 groups in GAIT continued to receive glucosamine 500 mg 3 times daily, CS 400 mg 3 times daily, the combination of glucosamine and CS, celecoxib 200 mg daily, or placebo over 24 months. The primary outcome measure was the number who reached a 20% reduction in WOMAC pain over 24 months. Secondary outcomes included reaching an OMERACT/OARSI response and change from baseline in WOMAC pain and function. Results The odds of achieving a 20%WOMAC were 1.21 for celecoxib, 1.16 for glucosamine, 0.83 for glucosamine and chondroitin sulfate and 0.69 for chondroitin sulfate alone with widely overlapping confidence intervals for all treatments. Conclusions Over 2 years, no treatment achieved a clinically important difference in WOMAC Pain or Function as compared with placebo. However, glucosamine and celecoxib showed beneficial trends. Adverse reactions were not meaningfully different among treatment groups and serious adverse events were rare for all therapies. PMID:20525840

Interface-Limited Spherulitic Growth of Hydroxyapatite/Chondroitin Sulfate Composite Enamel-like Films

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Ma, Guobin; Xu, Yifei; Wang, Xiyan; Wang, Mu

2012-02-01

Understanding and mimicking the growth of hard tissues such as tooth enamel may lead to innovative approaches toward engineering novel functional materials and providing new therapeutics. Up to now, in vitro growth of enamel-like materials is still a great challenge, and the microscopic formation mechanisms are far from well understood. Here we report synthesis of large-scale hydroxyapatite (HAP) and chondroitin sulfate (ChS) composite films by an efficient solution-air interface growth method. The products have the characteristic hierarchical prism structures of enamel and the mechanical properties comparable to dentin. We demonstrate that the films are assembled by spherulites nucleated at the solution surface. The growth of the spherulites is limited by the interfaces between them as well as between the solution and air, leading to the ordered prism structure. The results are beneficial for a clearer understanding of the fundamentals of tooth enamel formation.

The hidden Niemann-Pick type C patient: clinical niches for a rare inherited metabolic disease.

Science.gov (United States)

Hendriksz, Christian J; Anheim, Mathieu; Bauer, Peter; Bonnot, Olivier; Chakrapani, Anupam; Corvol, Jean-Christophe; de Koning, Tom J; Degtyareva, Anna; Dionisi-Vici, Carlo; Doss, Sarah; Duning, Thomas; Giunti, Paola; Iodice, Rosa; Johnston, Tracy; Kelly, Dierdre; Klünemann, Hans-Hermann; Lorenzl, Stefan; Padovani, Alessandro; Pocovi, Miguel; Synofzik, Matthis; Terblanche, Alta; Then Bergh, Florian; Topçu, Meral; Tranchant, Christine; Walterfang, Mark; Velten, Christian; Kolb, Stefan A

2017-05-01

Niemann-Pick disease type C (NP-C) is a rare, inherited neurodegenerative disease of impaired intracellular lipid trafficking. Clinical symptoms are highly heterogeneous, including neurological, visceral, or psychiatric manifestations. The incidence of NP-C is under-estimated due to under-recognition or misdiagnosis across a wide range of medical fields. New screening and diagnostic methods provide an opportunity to improve detection of unrecognized cases in clinical sub-populations associated with a higher risk of NP-C. Patients in these at-risk groups ("clinical niches") have symptoms that are potentially related to NP-C, but go unrecognized due to other, more prevalent clinical features, and lack of awareness regarding underlying metabolic causes. Twelve potential clinical niches identified by clinical experts were evaluated based on a comprehensive, non-systematic review of literature published to date. Relevant publications were identified by targeted literature searches of EMBASE and PubMed using key search terms specific to each niche. Articles published in English or other European languages up to 2016 were included. Several niches were found to be relevant based on available data: movement disorders (early-onset ataxia and dystonia), organic psychosis, early-onset cholestasis/(hepato)splenomegaly, cases with relevant antenatal findings or fetal abnormalities, and patients affected by family history, consanguinity, and endogamy. Potentially relevant niches requiring further supportive data included: early-onset cognitive decline, frontotemporal dementia, parkinsonism, and chronic inflammatory CNS disease. There was relatively weak evidence to suggest amyotrophic lateral sclerosis or progressive supranuclear gaze palsy as potential niches. Several clinical niches have been identified that harbor patients at increased risk of NP-C.

N-glycolyl groups of nonhuman chondroitin sulfates survive in ancient fossils.

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Bergfeld, Anne K; Lawrence, Roger; Diaz, Sandra L; Pearce, Oliver M T; Ghaderi, Darius; Gagneux, Pascal; Leakey, Meave G; Varki, Ajit

2017-09-26

Biosynthesis of the common mammalian sialic acid N -glycolylneuraminic acid (Neu5Gc) was lost during human evolution due to inactivation of the CMAH gene, possibly expediting divergence of the Homo lineage, due to a partial fertility barrier. Neu5Gc catabolism generates N -glycolylhexosamines, which are potential precursors for glycoconjugate biosynthesis. We carried out metabolic labeling experiments and studies of mice with human-like Neu5Gc deficiency to show that Neu5Gc degradation is the metabolic source of UDP-GlcNGc and UDP-GalNGc and the latter allows an unexpectedly selective incorporation of N -glycolyl groups into chondroitin sulfate (CS) over other potential glycoconjugate products. Partially N -glycolylated-CS was chemically synthesized as a standard for mass spectrometry to confirm its natural occurrence. Much lower amounts of GalNGc in human CS can apparently be derived from Neu5Gc-containing foods, a finding confirmed by feeding Neu5Gc-rich chow to human-like Neu5Gc-deficient mice. Unlike the case with Neu5Gc, N -glycolyl-CS was also stable enough to be detectable in animal fossils as old as 4 My. This work opens the door for investigating the biological and immunological significance of this glycosaminoglycan modification and for an "ancient glycans" approach to dating of Neu5Gc loss during the evolution of Homo .

Defect in dermatan sulfate in urine of patients with Ehlers-Danlos syndrome caused by a CHST14/D4ST1 deficiency.

Science.gov (United States)

Mizumoto, Shuji; Kosho, Tomoki; Hatamochi, Atsushi; Honda, Tomoko; Yamaguchi, Tomomi; Okamoto, Nobuhiko; Miyake, Noriko; Yamada, Shuhei; Sugahara, Kazuyuki

2017-08-01

Dermatan sulfate (DS) plays a number of roles in a wide range of biological activities such as cell signaling and tissue morphogenesis through interactions with various extracellular matrix proteins including collagen. Mutations in the carbohydrate sulfotransferase 14 gene (CHST14) encoding CHST14/dermatan 4-O-sulfotransferase-1 (D4ST1), which is responsible for the biosynthesis of DS, cause a recently delineated form of Ehlers-Danlos syndrome (EDS, musculocontractural type 1), an autosomal recessive connective tissue disorder characterized by congenital malformations (specific craniofacial features, and congenital multiple contractures) and progressive fragility-related complications (skin hyperextensibility, bruisability, and fragility with atrophic scars; recurrent dislocations; progressive talipes or spinal deformities; and large subcutaneous hematomas). In an attempt to develop a diagnostic screening method for this type of EDS, the amount of DS in the urine of patients was analyzed. Urinary DS was quantified by an anion-exchange chromatography after treatment with DS-specific degrading enzyme. DS was not detected in the urine of patients with homo- or compound heterozygous mutations in CHST14. These results suggest that the quantification of DS in urine is applicable to an initial diagnosis of DS-defective EDS. This is the first study to perform a urinary disaccharide compositional analysis of chondroitin sulfate (CS)/DS chains in patients with EDS caused by a CHST14/D4ST1 deficiency, and demonstrated the absence of DS chains. This result suggests systemic DS depletion in this disorder, and also proposes the usefulness of a urinary disaccharide compositional analysis of CS/DS chains as a non-invasive screening method for this disorder. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase.

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Wu, Mingyi; Wen, Dandan; Gao, Na; Xiao, Chuang; Yang, Lian; Xu, Li; Lian, Wu; Peng, Wenlie; Jiang, Jianmin; Zhao, Jinhua

2015-03-06

Fucosylated chondroitin sulfate (FCS), a structurally unusual glycosaminoglycan, has distinct anticoagulant properties, and is an especially strong inhibitor of the intrinsic factor Xase (anti-Xase). To obtain a highly selective inhibitor of human Xase, we purified six native FCSs with various sulfation patterns, prepared a series of FCS derivatives, and then elucidated the relationship between the structures and the anticoagulant activities of FCSs. FCSs 1-3 containing higher Fuc2S4S exhibit stronger AT-dependent anti-IIa activities, whereas 4-6 containing more Fuc3S4S produce potent HCII-dependent anti-IIa activities. Saccharides containing a minimum of 6-8 trisaccharide units, free carboxyl groups, and full fucosylation of GlcA may be required for potent anti-Xase activity, and approximately six trisaccharide units and partial fucosylation of GlcA may contribute to potent HCII-dependent activity. Decreasing of the molecular weights markedly reduces their AT-dependent anti-IIa activities, and even eliminates human platelet and factor XII activation. Furthermore, in vitro and in vivo studies suggested that fractions of 6-12 kDa may be very promising compounds as putative selective intrinsic Xase inhibitors with antithrombotic action, but without the consequences of major bleeding and factor XII activation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts

International Nuclear Information System (INIS)

Liscum, L.; Ruggiero, R.M.; Faust, J.R.

1989-01-01

Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol

Simultaneous analysis of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan disaccharides by glycoblotting-assisted sample preparation followed by single-step zwitter-ionic-hydrophilic interaction chromatography.

Science.gov (United States)

Takegawa, Yasuhiro; Araki, Kayo; Fujitani, Naoki; Furukawa, Jun-ichi; Sugiyama, Hiroaki; Sakai, Hideaki; Shinohara, Yasuro

2011-12-15

Glycosaminoglycans (GAGs) play important roles in cell adhesion and growth, maintenance of extracellular matrix (ECM) integrity, and signal transduction. To fully understand the biological functions of GAGs, there is a growing need for sensitive, rapid, and quantitative analysis of GAGs. The present work describes a novel analytical technique that enables high throughput cellular/tissue glycosaminoglycomics for all three families of uronic acid-containing GAGs, hyaluronan (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), and heparan sulfate (HS). A one-pot purification and labeling procedure for GAG Δ-disaccharides was established by chemo-selective ligation of disaccharides onto high density hydrazide beads (glycoblotting) and subsequent labeling by fluorescence. The 17 most common disaccharides (eight comprising HS, eight CS/DS, and one comprising HA) could be separated with a single chromatography for the first time by employing a zwitter-ionic type of hydrophilic-interaction chromatography column. These novel analytical techniques were able to precisely characterize the glycosaminoglycome in various cell types including embryonal carcinoma cells and ocular epithelial tissues (cornea, conjunctiva, and limbus).

Layer-by-layer assembly of type I collagen and chondroitin sulfate on aminolyzed PU for potential cartilage tissue engineering application

Energy Technology Data Exchange (ETDEWEB)

He Xianyun [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Wang Yingjun, E-mail: imwangyj@163.com [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China) and National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China) and Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Wu Gang, E-mail: imwugang@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China)

2012-10-01

Highlights: Black-Right-Pointing-Pointer A novel biodegradable polyurethane (PU) was successfully synthesized. Black-Right-Pointing-Pointer Surface aminolyzing of the PU was performed by reacting it with 1,3-propanediamine. Black-Right-Pointing-Pointer Collagen and chondroitin sulfate were deposited alternately on the PU surface. - Abstract: In this paper, a two-step method was used to synthesize a biodegradable polyurethane (PU) composed of L-lysine ethyl ester diisocyanate (LDI), poly({epsilon}-caprolactone) diols (PCL-diol) and 1,4:3,6-dianhydro-D-sorbitol (isosorbide). Amino groups were introduced onto the surface of the PU membrane by an amination reacting with 1,3-propanediamine to produce polycationic substratum. And then, type I collagen (Col) and chondroitin sulfate (CS) were deposited alternately on the polycationic substratum through layer-by-layer (LBL) assembly technology. The FTIR and {sup 1}H NMR results showed that the polyurethane was successfully synthesized. Rhodamine B isothiocyanate (RBITC) fluorescence spectrum indicated that amino groups were successfully introduced onto the PU surface. The results of quartz-crystal microbalance (QCM) and RBITC-Col fluorescence spectroscopy monitoring the LBL assemble process presented that the Col/CS deposited alternately on the PU surface. X-ray photoelectron spectroscopy (XPS) results displayed that the CS deposited on the PU surface as well. The surface of the assembled PU became even smoother observed from the surface morphology by atomic force microscopy (AFM) imaging. The hydrophilicity of the PU membrane was greatly enhanced though the modification of LBL assembly. The PU modified with the adsorption of Col/CS may be a potential application for cartilage tissue engineering due to its created mimicking chondrogenic environment.

Chondroitin sulfate and sodium hialuronate in treatment of the degenerative joint disease in dogs. Clinical and radiological aspects

International Nuclear Information System (INIS)

Melo, E.G.; Rezende, C.M.F.; Gomes, M.G.; Freitas, P.M.; Arias, S.S.A.

2003-01-01

The aim of this study was the evaluation of the effects of chondroitin sulfate and sodium hyaluronate in the knee joint of dogs with experimentally induced degenerative joint disease (DJD). Fifteen mongrel dogs, weighing 18 to 25kg were used. DJD was induced by cranial cruciate ligament (CCL) arthroscopical transection. After three weeks, CCL was repaired by an intrarticular technique, which uses fascia lata. The dogs were then divided into three groups as follows: group I received no other treatment, but the CCL reconstitution, group II received 24mg/animal of chondroitin sulfate/IM every five days, totaling six injections, and group III received 20mg/animal of sodium hyaluronate /IV every five days, totaling three injections. All dogs were examined clinically and radiographically for 90 days after the repairment surgery. The clinical evaluation was performed by assessment of lameness, weight-bearing, limb muscle atrophy and range of motion. The results demonstrated that the group treated with sodium hyaluronate had lower degree of lameness in comparison with other groups. The radiographic evaluation showed marginal osteophytes and subchondral bone sclerosis. These changes were more severe in the group treated with sodium hyaluronate. The better clinical results observed in this group, compared with the others, was probably due to the greater action of the drug in the synovium, decreasing the pain and lameness. Radiographic findings correlated poorly with the clinical signs in the group treated with sodium hyaluronate [pt

Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

DEFF Research Database (Denmark)

Maccarana, M.; Kalamajski, S.; Kongsgaard, M.

2009-01-01

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks......-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found...... that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile...

Analysis of oversulfation in biglycan chondroitin/dermatan sulfate oligosaccharides by chip-based nanoelectrospray ionization multistage mass spectrometry.

Science.gov (United States)

Flangea, Corina; Sisu, Eugen; Seidler, Daniela G; Zamfir, Alina D

2012-01-15

Biglycan (BGN) is a small proteoglycan that consists of a protein core containing leucine-rich repeat regions and two glycosaminoglycan (GAG) chains of either chondroitin sulfate (CS) or dermatan sulfate (DS) type. The development of novel, highly efficient analytical methods for structural identification of BGN-derived CS/DS motifs, possibly implicated in biological events, is currently the focus of research. In this work, an improved analytical method based on fully automated chip-nanoelectrospray ionization (nanoESI) in conjunction with high-capacity ion trap (HCT) multistage mass spectrometry (MS) by collision-induced dissociation (CID) was for the first time applied to BGN CS/DS oligosaccharide analysis. The CS/DS chains were released from transfected 293 BGN by β-elimination. The chain was digested with AC I lyase, and the resulting mixture was purified and subsequently separated by size exclusion chromatography (SEC). Di- and tetrasaccharide fractions were pooled and characterized in detail using the developed chip-nanoESI protocol. The chip-nanoESI MS profile in the negative ion mode revealed the presence of under-, regularly, and oversulfated species in both di- and tetrasaccharide fractions. CID MS(2)-MS(3) yielded sequence patterns consistent with unusual oversulfated 4,5-Δ-GlcA(2S)-GalNAc(4S) and 4,5-Δ-GlcA(2S)-GalNAc(6S)-IdoA(2S)-GalNAc(6S) motifs. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro fermentation behaviors of fucosylated chondroitin sulfate from Pearsonothuria graeffei by human gut microflora.

Science.gov (United States)

Wei, Chao-Yang; Liao, Ning-Bo; Zhang, Yu; Ye, Xing-Qian; Li, Shan; Hu, Ya-Qin; Liu, Dong-Hong; Linhardt, Robert J; Wang, Xin; Chen, Shi-Guo

2017-09-01

A fucosylated chondroitin sulfate (FCS-pg) with highly repeated structure from Pearsonothuria graeffei was subjected to a in vitro fermentation model to investigate its fermentability and effects on human gut microflora. High performance liquid chromatography (HPLC) measurement found FCS-pg can be fermented to short chain fatty acids (SCFAs) by gut microflora from partial human fecal samples. 16S rRNA gene-based polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) profiling and real-time quantitative PCR analysis showed that FCS-pg mainly increased the proportions of Clostridium cluster XI, Bacteriodes prevotella group, Bifidobacterium genus, Clostridium cluster I and Clostridium cluster XIVab, whereas the numbers of the Enterobacteriaceae and Lactobacillus decreased. These results indicated that FCS-pg was mainly fermented by Bacteroides, Bifidobacterium and Clostridium. It increased the content of probiotics bacteria in achieving health-enhancing effect, was slightly different than most sulfated polysaccharides from marine animals. The current study provides useful new information on the mechanism of absorption and functional activity on FCS-pg within the gastrointestinal tract of the human body. Copyright © 2017. Published by Elsevier B.V.

A Preliminary Analysis of the Phenomenology of Skin-Picking in Prader-Willi Syndrome

Science.gov (United States)

Morgan, Jessica R.; Storch, Eric A.; Woods, Douglas W.; Bodzin, Danielle; Lewin, Adam B.; Murphy, Tanya K.

2010-01-01

To examine the nature and psychosocial correlates of skin-picking behavior in youth with Prader-Willi Syndrome (PWS). Parents of 67 youth (aged 5-19 years) with PWS were recruited to complete an internet-based survey that included measures of: skin-picking behaviors, the automatic and/or focused nature of skin-picking, severity of skin-picking…

Novel Insights into Antiviral Gene Regulation of Red Swamp Crayfish, Procambarus clarkii, Infected with White Spot Syndrome Virus

Directory of Open Access Journals (Sweden)

Shaokui Yi

2017-11-01

Full Text Available White spot syndrome virus (WSSV, one of the major pathogens of Procambarus clarkii, has caused severe disruption to the aquaculture industry of P. clarkii in China. To reveal the gene regulatory mechanisms underlying WSSV infection, a comparative transcriptome analysis was performed among WSSV-infected susceptible individuals (GS, viral resistant individuals (GR, and a non-infected control group (GC. A total of 61,349 unigenes were assembled from nine libraries. Subsequently, 515 and 1033 unigenes exhibited significant differential expression in sensitive and resistant crayfish individuals compared to the control group (GC. Many differentially expressed genes (e.g., C-type lectin 4, Peroxinectin, Prophenoloxidase, and Serine/threonine-protein kinase observed in GR and GS play critical roles in pathogen recognition and viral defense reactions after WSSV infection. Importantly, the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate pathway was identified to play critical roles in defense to WSSV infection for resistant crayfish individuals by upregulating the chondroitin sulfate related genes for the synthesis of WSSV-sensitive, functional chondroitin sulfate chains containing E units. Numerous genes and the key pathways identified between resistant and susceptible P. clarkii individuals provide valuable insights regarding antiviral response mechanisms of decapoda species and may help to improve the selective breeding of P. clarkii WSSV-resistance.

Improved anticoagulant effect of fucosylated chondroitin sulfate orally administered as gastro-resistant tablets.

Science.gov (United States)

Fonseca, Roberto J C; Sucupira, Isabela D; Oliveira, Stephan Nicollas M C G; Santos, Gustavo R C; Mourão, Paulo A S

2017-04-03

Fucosylated chondroitin sulfate (FucCS) is a potent anticoagulant polysaccharide extracted from sea cucumber. Its anticoagulant activity is attributed to the presence of unique branches of sulfated fucose. Although this glycosaminoglycan exerts an antithrombotic effect following oral administration, high doses are necessary to achieve the maximum effect. The diminished activity of FucCS following oral administration is likely due to its degradation in the gastrointestinal tract and its limited ability to cross the intestinal cell membranes. The latter aspect is particularly difficult to overcome. However, gastro-resistant tablet formulation may help limit the degradation of FucCS in the gastrointestinal tract. In the present work, we found that the oral administration of FucCS as gastro-resistant tablets produces a more potent and prolonged anticoagulant effect compared with its administration as an aqueous solution, with no significant changes in the bleeding tendency or arterial blood pressure. Experiments using animal models of arterial thrombosis initiated by endothelial injury demonstrated that FucCS delivered as gastro-protective tablets produced a potent antithrombotic effect, whereas its aqueous solution was ineffective. However, there was no significant difference between the effects of FucCS delivered as gastro-resistant tablets or as aqueous solution in a venous thrombosis model, likely due to the high dose of thromboplastin used. New oral anticoagulants tested in these experimental models for comparison showed significantly increased bleeding tendencies. Our study provides a framework for developing effective oral anticoagulants based on sulfated polysaccharides from marine organisms. The present results suggest that FucCS is a promising oral anticoagulant.

Chondroitin sulfate-capped super-paramagnetic iron oxide nanoparticles as potential carriers of doxorubicin hydrochloride.

Science.gov (United States)

Mallick, Neha; Anwar, Mohammed; Asfer, Mohammed; Mehdi, Syed Hassan; Rizvi, Mohammed Moshahid Alam; Panda, Amulya Kumar; Talegaonkar, Sushama; Ahmad, Farhan Jalees

2016-10-20

Chondroitin-4-sulfate (CS), a glycosaminoglycan, was used to prepare CS-capped super-paramagnetic iron oxide nanoparticles, which were further employed for loading a water-soluble chemotherapeutic agent (doxorubicin hydrochloride, DOX). CS-capped SPIONs have potential biomedical application in cancer targeting. The optimized formulation had a hydrodynamic size of 91.2±0.8nm (PDI; 0.228±0.004) and zeta potential of -49.1±1.66mV. DOX was loaded onto the formulation up to 2% (w/w) by physical interaction with CS. TEM showed nano-sized particles having a core-shell structure. XRD confirmed crystal phase of iron oxide. FT-IR conceived the interaction of iron oxide with CS as bidentate chelation and also confirmed DOX loading. Vibration sample magnetometry confirmed super-paramagnetic nature of nanoparticles, with saturation magnetization of 0.238emug(-1). In vitro release profile at pH 7.4 showed that 96.67% of DOX was released within 24h (first order kinetics). MTT assay in MCF7 cells showed significantly higher (p<0.0001) cytotoxicity for DOX in SPIONs than DOX solution (IC50 values 6.294±0.4169 and 11.316±0.1102μgmL(-1), respectively). Copyright © 2016 Elsevier Ltd. All rights reserved.

Delivery of acid sphingomyelinase in normal and niemann-pick disease mice using intercellular adhesion molecule-1-targeted polymer nanocarriers.

Science.gov (United States)

Garnacho, Carmen; Dhami, Rajwinder; Simone, Eric; Dziubla, Thomas; Leferovich, John; Schuchman, Edward H; Muzykantov, Vladimir; Muro, Silvia

2008-05-01

Type B Niemann-Pick disease (NPD) is a multiorgan system disorder caused by a genetic deficiency of acid sphingomyelinase (ASM), for which lung is an important and challenging therapeutic target. In this study, we designed and evaluated new delivery vehicles for enzyme replacement therapy of type B NPD, consisting of polystyrene and poly(lactic-coglycolic) acid polymer nanocarriers targeted to intercellular adhesion molecule (ICAM)-1, an endothelial surface protein up-regulated in many pathologies, including type B NPD. Real-time vascular imaging using intravital microscopy and postmortem imaging of mouse organs showed rapid, uniform, and efficient binding of fluorescently labeled ICAM-1-targeted ASM nanocarriers (anti-ICAM/ASM nanocarriers) to endothelium after i.v. injection in mice. Fluorescence microscopy of lung alveoli actin, tissue histology, and 125I-albumin blood-to-lung transport showed that anti-ICAM nanocarriers cause neither detectable lung injury, nor abnormal vascular permeability in animals. Radioisotope tracing showed rapid disappearance from the circulation and enhanced accumulation of anti-ICAM/125I-ASM nanocarriers over the nontargeted naked enzyme in kidney, heart, liver, spleen, and primarily lung, both in wild-type and ASM knockout mice. These data demonstrate that ICAM-1-targeted nanocarriers may enhance enzyme replacement therapy for type B NPD and perhaps other lysosomal storage disorders.

Trafficking of cholesterol from cell bodies to distal axons in Niemann Pick C1-deficient neurons.

Science.gov (United States)

Karten, Barbara; Vance, Dennis E; Campenot, Robert B; Vance, Jean E

2003-02-07

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disorder. In cells lacking functional NPC1 protein, endocytosed cholesterol accumulates in late endosomes/lysosomes. We utilized primary neuronal cultures in which cell bodies and distal axons reside in separate compartments to investigate the requirement of NPC1 protein for transport of cholesterol from cell bodies to distal axons. We have recently observed that in NPC1-deficient neurons compared with wild-type neurons, cholesterol accumulates in cell bodies but is reduced in distal axons (Karten, B., Vance, D. E., Campenot, R. B., and Vance, J. E. (2002) J. Neurochem. 83, 1154-1163). We now show that NPC1 protein is expressed in both cell bodies and distal axons. In NPC1-deficient neurons, cholesterol delivered to cell bodies from low density lipoproteins (LDLs), high density lipoproteins, or cyclodextrin complexes was transported into axons in normal amounts, whereas transport of endogenously synthesized cholesterol was impaired. Inhibition of cholesterol synthesis with pravastatin in wild-type and NPC1-deficient neurons reduced axonal growth. However, LDLs restored a normal rate of growth to wild-type but not NPC1-deficient neurons treated with pravastatin. Thus, although LDL cholesterol is transported into axons of NPC1-deficient neurons, this source of cholesterol does not sustain normal axonal growth. Over the lifespan of NPC1-deficient neurons, these defects in cholesterol transport might be responsible for the observed altered distribution of cholesterol between cell bodies and axons and, consequently, might contribute to the neurological dysfunction in NPC disease.

Lycopene reduces cholesterol absorption through the downregulation of Niemann-Pick C1-like 1 in Caco-2 cells.

Science.gov (United States)

Zou, Jun; Feng, Dan

2015-11-01

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Tomato lycopene has been found to have a hypocholesterolemic effect, and the effect was considered to be related to inhibition of cholesterol synthesis. However, since plasma cholesterol levels are also influenced by the absorption of cholesterol in the gut, the present study is to investigate whether lycopene affects cholesterol absorption in the intestinal Caco-2 cells. The Caco-2 cells were pretreated with lycopene at different concentrations for 24 h and then incubated with radioactive micellar cholesterol for 2 h. The absorption of radioactive cholesterol was quantified by liquid scintillation. The expression of Niemann-Pick C1-like 1 (NPC1L1) and liver X receptor α (LXRα) was analyzed by Western blot and qPCR. We found that lycopene dose dependently inhibited cholesterol absorption and the expression of NPC1L1 protein and NPC1L1 mRNA. The inhibitory effects of lycopene on cholesterol absorption and NPC1L1 expression could be prevented by blockade of the LXRα pathway. This study provides the first evidence that lycopene inhibits cholesterol absorption in the intestinal cells and this inhibitory effect of lycopene is mediated, at least in part, by LXRα-NPC1L1 signaling pathway. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PEG-lipid micelles enable cholesterol efflux in Niemann-Pick Type C1 disease-based lysosomal storage disorder

Science.gov (United States)

Brown, Anna; Patel, Siddharth; Ward, Carl; Lorenz, Anna; Ortiz, Mauren; Duross, Allison; Wieghardt, Fabian; Esch, Amanda; Otten, Elsje G.; Heiser, Laura M.; Korolchuk, Viktor I.; Sun, Conroy; Sarkar, Sovan; Sahay, Gaurav

2016-08-01

2-Hydroxy-propyl-β-cyclodextrin (HPβCD), a cholesterol scavenger, is currently undergoing Phase 2b/3 clinical trial for treatment of Niemann Pick Type C-1 (NPC1), a fatal neurodegenerative disorder that stems from abnormal cholesterol accumulation in the endo/lysosomes. Unfortunately, the extremely high doses of HPβCD required to prevent progressive neurodegeneration exacerbates ototoxicity, pulmonary toxicity and autophagy-based cellular defects. We present unexpected evidence that a poly (ethylene glycol) (PEG)-lipid conjugate enables cholesterol clearance from endo/lysosomes of Npc1 mutant (Npc1-/-) cells. Herein, we show that distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), which forms 12-nm micelles above the critical micelle concentration, accumulates heavily inside cholesterol-rich late endosomes in Npc1-/- cells. This potentially results in cholesterol solubilization and leakage from lysosomes. High-throughput screening revealed that DSPE-PEG, in combination with HPβCD, acts synergistically to efflux cholesterol without significantly aggravating autophagy defects. These well-known excipients can be used as admixtures to treat NPC1 disorder. Increasing PEG chain lengths from 350 Da-30 kDa in DSPE-PEG micelles, or increasing DSPE-PEG content in an array of liposomes packaged with HPβCD, improved cholesterol egress, while Pluronic block copolymers capable of micelle formation showed slight effects at high concentrations. We postulate that PEG-lipid based nanocarriers can serve as bioactive drug delivery systems for effective treatment of lysosomal storage disorders.

PEG-lipid micelles enable cholesterol efflux in Niemann-Pick Type C1 disease-based lysosomal storage disorder

Science.gov (United States)

Brown, Anna; Patel, Siddharth; Ward, Carl; Lorenz, Anna; Ortiz, Mauren; DuRoss, Allison; Wieghardt, Fabian; Esch, Amanda; Otten, Elsje G.; Heiser, Laura M.; Korolchuk, Viktor I.; Sun, Conroy; Sarkar, Sovan; Sahay, Gaurav

2016-01-01

2-Hydroxy-propyl-β-cyclodextrin (HPβCD), a cholesterol scavenger, is currently undergoing Phase 2b/3 clinical trial for treatment of Niemann Pick Type C-1 (NPC1), a fatal neurodegenerative disorder that stems from abnormal cholesterol accumulation in the endo/lysosomes. Unfortunately, the extremely high doses of HPβCD required to prevent progressive neurodegeneration exacerbates ototoxicity, pulmonary toxicity and autophagy-based cellular defects. We present unexpected evidence that a poly (ethylene glycol) (PEG)-lipid conjugate enables cholesterol clearance from endo/lysosomes of Npc1 mutant (Npc1−/−) cells. Herein, we show that distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), which forms 12-nm micelles above the critical micelle concentration, accumulates heavily inside cholesterol-rich late endosomes in Npc1−/− cells. This potentially results in cholesterol solubilization and leakage from lysosomes. High-throughput screening revealed that DSPE-PEG, in combination with HPβCD, acts synergistically to efflux cholesterol without significantly aggravating autophagy defects. These well-known excipients can be used as admixtures to treat NPC1 disorder. Increasing PEG chain lengths from 350 Da-30 kDa in DSPE-PEG micelles, or increasing DSPE-PEG content in an array of liposomes packaged with HPβCD, improved cholesterol egress, while Pluronic block copolymers capable of micelle formation showed slight effects at high concentrations. We postulate that PEG-lipid based nanocarriers can serve as bioactive drug delivery systems for effective treatment of lysosomal storage disorders. PMID:27572704

Role of Diffusion Tensor Imaging in Prognostication and Treatment Monitoring in Niemann-Pick Disease Type C1

Directory of Open Access Journals (Sweden)

Meghann W. Lau

2016-09-01

Full Text Available Niemann-Pick Disease, type C1 (NPC1 is a rapidly progressive neurodegenerative disorder characterized by cholesterol sequestration within late endosomes and lysosomes, for which no reliable imaging marker exists for prognostication and management. Cerebellar volume deficits are found to correlate with disease severity and diffusion tensor imaging (DTI of the corpus callosum and brainstem, which has shown that microstructural disorganization is associated with NPC1 severity. This study investigates the utility of cerebellar DTI in clinical severity assessment. We hypothesize that cerebellar volume, fractional anisotropy (FA and mean diffusivity (MD negatively correlate with NIH NPC neurological severity score (NNSS and motor severity subscores. Magnetic resonance imaging (MRI was obtained for thirty-nine NPC1 subjects, ages 1–21.9 years (mean = 11.1, SD = 6.1. Using an atlas-based automated approach, the cerebellum of each patient was measured for FA, MD and volume. Additionally, each patient was given an NNSS. Decreased cerebellar FA and volume, and elevated MD correlate with higher NNSS. The cognition subscore and motor subscores for eye movement, ambulation, speech, swallowing, and fine motor skills were also statistically significant. Microstructural disorganization negatively correlated with motor severity in subjects. Additionally, Miglustat therapy correlated with lower severity scores across ranges of FA, MD and volume in all regions except the inferior peduncle, where a paradoxical effect was observed at high FA values. These findings suggest that DTI is a promising prognostication tool.

Correlation between chondroitin sulfate iron colloid - enhanced MR imaging and the histological grade of hepatocellular carcinoma

Energy Technology Data Exchange (ETDEWEB)

Suto, Y. [Dept. of Radiology, Tottori University School of Medcine, Yonago (Japan); Kodama, F. [Dept. of Radiology, Tottori University School of Medcine, Yonago (Japan); Kamba, M. [Dept. of Radiology, Tottori University School of Medcine, Yonago (Japan); Ohta, Y. [Dept. of Radiology, Tottori University School of Medcine, Yonago (Japan)

1995-01-01

The association between contrast enhancement by chondroitin sulfate iron colloid (CSIC) and the histological grade of hepatocellular carcinoma (HCC) was evaluated in 24 patients diagnosed by histological examination of surgical specimens (26 nodules: 11 well-differentiated and 15 poorly-moderately-differentiated nodules). In the well-differentiated HCC nodules, the tumor-liver contrast to noise ratio (CNR) was not significantly increased after i.v. CSIC injection on both T1-weighted and T2-weighted images. In the moderately-poorly-differentiated HCC, CNR was significantly increased after CSIC administration on both T1-weighted and T2-weighted images (p<0.01). MR imaging using CSIC may be useful for diagnosing the degree of HCC differentiation. (orig.).

Correlation between chondroitin sulfate iron colloid - enhanced MR imaging and the histological grade of hepatocellular carcinoma

International Nuclear Information System (INIS)

Suto, Y.; Kodama, F.; Kamba, M.; Ohta, Y.

1995-01-01

The association between contrast enhancement by chondroitin sulfate iron colloid (CSIC) and the histological grade of hepatocellular carcinoma (HCC) was evaluated in 24 patients diagnosed by histological examination of surgical specimens (26 nodules: 11 well-differentiated and 15 poorly-moderately-differentiated nodules). In the well-differentiated HCC nodules, the tumor-liver contrast to noise ratio (CNR) was not significantly increased after i.v. CSIC injection on both T1-weighted and T2-weighted images. In the moderately-poorly-differentiated HCC, CNR was significantly increased after CSIC administration on both T1-weighted and T2-weighted images (p<0.01). MR imaging using CSIC may be useful for diagnosing the degree of HCC differentiation. (orig.)

Crystallization of calcium oxalate monohydrate at dipalmitoylphosphatidylcholine monolayers in the presence of chondroitin sulfate A

Science.gov (United States)

Ouyang, Jian-Ming; Deng, Sui-Ping; Zhong, Jiu-Ping; Tieke, Bernd; Yu, Shu-Hong

2004-10-01

The growth and aggregation of calcium oxalate monohydrate (COM) crystals beneath dipalmitoylphosphatidylcholine (DPPC) monolayers in the presence of chondroitin sulfate A (C4S) was systematically examined under different surface pressure. The results indicated that the addition of C4S can inhibit the crystal growth and prevent the aggregation of COM crystals. Under a DPPC monolayer, well-defined three-dimensional hexagonal prisms and three-dimensional rhombus prisms with sharply angled tips were obtained. The DPPC monolayer at a surface pressure of 10 mN/m can match the Ca2+ distance of the (1 bar 0 1) face of COM better than at 20 mN/m. The addition of C4S could cooperatively modulate the interaction strength between the monolayer (or itself) with the specific morphology determining faces such as (1 bar 0 1) and (0 2 0), and thus results in remarkable stabilization of the (1 bar 0 1) faces. The dramatic changes in morphological details were due to the strong electrostatic interactions between the Ca2+-rich (1 bar 0 1) crystal faces of COM and the polyanionic polysaccharide C4S together with the negatively charged sites of the zwitterionic DPPC monolayers. The increase of the concentration of C4S can further enhance the stabilization of the (1 bar 0 1) face.

In vivo gene transfer using pDNA/chitosan/chondroitin sulfate ternary complexes: influence of chondroitin sulfate on the stability of freeze-dried complexes and transgene expression in vivo.

Science.gov (United States)

Hagiwara, Kenji; Kishimoto, Satoko; Ishihara, Masayuki; Koyama, Yoshiyuki; Mazda, Osam; Sato, Toshinori

2013-02-01

Chitosan has been investigated as a promising nonviral vector. However, several problems still remain, such as a relatively low transfection efficiency and instability under physiological conditions. We previously demonstrated that a chondroitin sulfate (CS) coating enhanced the transfection efficiency and physicochemical stability of plasmid DNA (pDNA)/chitosan complexes in vitro. In the present study, the effects of coating pDNA/chitosan complexes with CS on the stability in freeze-dry rehydration processes and gene expression in vivo were investigated. Freeze-drying storage at -20 °C, 4 °C, or room temperature, freezing storage at -20 °C, or liquid storage at 4 °C or room temperature, were examined for preservation conditions of pDNA/chitosan/CS ternary complexes by a gel retardation assay, measurements of sizes and zeta potentials, and a luciferase assay. Moreover, to determine the transfection efficiency of the ternary complexes in vivo, suicide gene therapy was carried out in Huh-7-implanted mice using herpes simplex virus thymidine kinase coding pDNA and ganciclovir. The freeze-dried pDNA/chitosan/CS ternary complexes showed sufficient cell transfection ability in vitro and in vivo. In addition, ternary complexes were associated with a significant suppression of tumor growth and a histopathologically high anti-tumor effect by intratumoral injection to tumor-bearing mice. The CS coating enhanced the preservation stability of the pDNA/chitosan complexes after freeze-drying-rehydration and their transgene expression in vivo. Copyright © 2013 John Wiley & Sons, Ltd.

About the singular behavior of the ionic condensation of sodium chondroitin sulfate: Conductivity study in water and water dioxane mixture

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M'halla, Jalel; Besbes, Rafik; Bouazzi, Ramzi; Boughammoura, Sondes

2006-01-01

In this work, we generalized the (Bjerrum-Debye-Fuoss-MSA) double layer model to an ellipsoidal polyion (chondroitin sulfate) of (∣ Zs∣ e) structural charge, Ls structural length, R minor axe and ( R2 + L2/4) 1/2 major axe. With L ⩽ Ls. Na + counter ions are distributed on the contact (or condensed) layer and on the Debye layer (ionic atmosphere). Both layers are ellipsoidal equipotentials of, respectively, R and d minor axes and are concentric to the polyion. With d = ( R + 1/2 Γ), Γ is the Debye-MSA screen parameter. The equilibrium distribution of Na + ions is derived from a "two states" statistical approach, leading to a general implicit expression for the rate of condensation (1 - α). The generality of this formula results from the fact that it takes into account the finite size of the polyion ( L ≠ ∞ and R ≠ 0) and allows to calculate α for different conformations of the polyion: (ellipsoidal L ≠ 0, cylindrical: L = Ls, spherical: L → 0, and Manning's model: RL-1 → 0). The main conclusion of this model is that, α obeys to the Ostwald's principle of dilution ( α → 1 when CNa+ → 0). This result is contrary to Manning's theory, for which α is a constant αM independent on the concentration Ci: αM = bS/(∣ Zi∣ Lb), with bS = Ls/∣ Zs∣ and Lb = e2/( ɛkT) is the Bjerrum length. However, our analysis shows that the rate of variation: (∂ α/∂ Ci) in a given range of concentration, depends on the structural parameter bS. Indeed, the critical Manning condition ( α-1αM = 1, ⇒(∂ α/∂ Ci) ≈ 0), is compatible with the general following "rod-like model" approximation: (1-α)≈|Zi|(πbS)[4πZi2Lb][αCiR2];withbS″=αM-1 only for some peculiar values of bS and Lb (i.e., dielectric constant: ɛ). In water at 25 °C ( ɛ = 78.3), this singular behavior occurs for a range of a relative low or moderate concentration for some polyelectrolytes of bS structural parameter of about 5.8 Å. This is the case of sodium chondroitin

Influence of charge on FITC-BSA-loaded chondroitin sulfate-chitosan nanoparticles upon cell uptake in human Caco-2 cell monolayers

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Hu CS

2012-09-01

Full Text Available Chieh-shen Hu,1 Chiao-hsi Chiang,2 Po-da Hong,1,4,* Ming-kung Yeh1–3,*1Biomedical Engineering Program, Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology; 2School of Pharmacy, National Defence Medical Center; 3Bureau of Pharmaceutical Affairs, Ministry of National Defence Medical Affairs Bureau; 4Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taiwan, Republic of China*These authors contributed equally to this workBackground and methods: Chondroitin sulfate-chitosan (ChS-CS nanoparticles and positively and negatively charged fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA-loaded ChS-CS nanoparticles were prepared and characterized. The properties of ChS-CS nanoparticles, including cellular uptake, cytotoxicity, and transepithelial transport, as well as findings on field emission-scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy were evaluated in human epithelial colorectal adenocarcinoma (Caco-2 fibroblasts. ChS-CS nanoparticles with a mean particle size of 250 nm and zeta potentials ranging from –30 to +18 mV were prepared using an ionic gelation method.Results: Standard cell viability assays demonstrated that cells incubated with ChS-CS and FITC-BSA-loaded ChS-CS nanoparticles remained more than 95% viable at particle concentrations up to 0.1 mg/mL. Endocytosis of nanoparticles was confirmed by confocal laser scanning microscopy and measured by flow cytometry. Ex vivo transepithelial transport studies using Caco-2 cells indicated that the nanoparticles were effectively transported into Caco-2 cells via endocytosis. The uptake of positively charged FITC-BSA-loaded ChS-CS nanoparticles across the epithelial membrane was more efficient than that of the negatively charged nanoparticles.Conclusion: The ChS-CS nanoparticles fabricated in this study were

Phase I/II Pilot Study of Mixed Chimerism to Treat Inherited Metabolic Disorders

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2017-11-15

Hurler Syndrome (MPS I); Hurler-Scheie Syndrome; Hunter Syndrome (MPS II); Sanfilippo Syndrome (MPS III); Krabbe Disease (Globoid Leukodystrophy); Metachromatic Leukodystrophy (MLD); Adrenoleukodystrophy (ALD and AMN); Sandhoff Disease; Tay Sachs Disease; Pelizaeus Merzbacher (PMD); Niemann-Pick Disease; Alpha-mannosidosis

99mTc-labeled chondroitin sulfate-uptake by chondrocytes and cartilage. Potential agent for osteoarthritis imaging?

International Nuclear Information System (INIS)

Sobal, G.; Sinzinger, H.; Menzel, J.

2002-01-01

Aim: Chondroitin sulfate (CS) is an endogenous component of cartilage proteoglycan which could monitor osteoarthritic cartilage degradation after radiolabeling. This substance is used in the treatment of human osteoarthritis as a slow acting symptomatic drug (CONDROSULF; Sanova Pharma, Vienna; Ibsa, Switzerland). Material and Methods: Radiolabeling of CS was performed using 99m TcO 4 -/stannous chloride in 0.50 M sodium acetate buffer at pH 5.0. The quality control of the tracer was performed using ITLC-SG chromatography and 0.2 M saline in 10% ethanol as solvent to detect colloid content. Aluminium oxide IB-F TLC-sheets and ethanol as solvent were used to estimate free pertechnetate. For uptake studies cultured human chondrocytes and age-matched cartilage were used. Uptake of the tracer in chondrocytes was studied in monolayer and in suspension cultures at 37 0 C. Uptake was monitored for a total of 120-180 minutes, samples being drawn every 10 minutes. Because the commercially available drug Condrosulf contains calcium stearate as additive to improve the resorption of the drug, we investigated also the uptake with and without additive. Results: The tracer was stable over 6h period after labeling (95% of the radiochemical purity). In plasma the stability was lower amounting to 75%. Viability of chondrocytes after incubation with either CS-preparation was found by trypan blue exclusion to be above 95 %. Uptake of the tracer performed in monolayer ± additives was low and amounted to 0.5%±0.05%, n=6. The cells were saturated already after an incubation interval of 10 minutes. In suspension cultures a maximal uptake of 1.0%±0.1%, n=6 and 5.9%±0.65%, n=6 was found, without and with additives, respectively, the saturation was achieved after 100 min. Thus, not only the resorption of the drug is enhanced by Ca-stearate, but also uptake increases in presence of this additive. Using human rib cartilage the uptake of the tracer was much higher amounting to 4.9%±2.3%, n

Synthesis and NMR analysis of model compounds related to fucosylated chondroitin sulfates: GalNAc and Fuc(1 → 6)GalNAc derivatives.

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Vinnitskiy, Dmitry Z; Ustyuzhanina, Nadezhda E; Dmitrenok, Andrey S; Shashkov, Alexander S; Nifantiev, Nikolay E

2017-01-13

Unsubstituted and 6-O-α-L-fucosylated propyl 2-acetamido-2-deoxy-β-D-galactopyranosides and their selectively O-sulfated (both in GalNAc and Fuc units) derivatives were synthesized as model compounds representing the fragments of fucosylated chondroitin sulfates (FCS) from sea cucumbers. Per-O-acetylated 2-deoxy-2-N-phthalimido-D-glucopyranose was used as a key precursor for the preparation of all 2-acetamido-2-deoxy-D-galactopyranoside containing products. Attempts at 6-O-glycosylation of propyl 3-O-benzoyl-2-deoxy-2-N-phthalimido-D-galactoside by 2-O-benzyl-3,4-di-O-chloracetyl-L-fucosyl trichloracetimidate in the presence of TMSOTf gave a 1:1 mixture of the corresponding α- and β-isomeric disaccharides, while the use of structurally related fucosyl bromide donor with promotion by Bu 4 NBr led to the formation of desired α-isomeric disaccharide exclusively. Selective removal of orthogonal O-protections permitted subsequent O-sulfation both at the GalNAc and Fuc units. Further removal of blocking groups yielded the target products which were systematically studied by 1 H and 13 C NMR spectroscopy in order to determine the spectral effects of O-sulfation and α-L-fucosylation needed for the development of computer assisted structural analysis of natural FCS. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis, characterization and application in biomedicine of a novel chondroitin sulfate based hydrogel and bioadhesive

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Strehin, Iossif

Clinically, there exists a need for adhesive biomaterials. There is room to improve upon what is currently on the market as it is either too toxic, lacks the required adhesive strength and/or lacks the desired degradation properties. The general goals of this thesis all focused on designing a biomaterial which would improve upon these shortcomings while at the same time allow for modifications to meet the needs for the specific application of interest. To accomplish this task, it was important to choose the appropriate composition and crosslinking chemistry which will allow the most flexibility. Chondroitin sulfate (CS) was chosen as the principle component of the hydrogel because it is a ubiquitous glycosaminoglycan (GAG) found in almost all tissues in the body. Many variants of CS exist with each one possessing unique biological activity allowing for tight control over these properties of the material. To modulate cell migration through the adhesive, polyethylene glycol (PEG) or blood was used as the second constituent. The former made the scaffold act as a cell barrier while the ladder could be used in varying concentrations to modulate cell adhesion and migration into the biomaterial. Also, the CS and blood components are both biodegradable and degradation can be controlled using various methods. While the constituents were chosen to allow flexibility in the biological activity and cell migration into the scaffold, the crosslinking chemistry was chosen to allow control over the mechanical properties as well as to increase tissue adhesion. By functionalizing the carboxyl groups of the GAG with N-hydroxysuccinimide (NHS), the resulting chondroitin sulfate succinimidyl succinate (CS-NHS) molecule could react with primary amines on polymers to form a hydrogel as well as the primary amines on proteins comprising tissue to anchor the hydrogel to the tissue. The material has been characterized and optimized for several applications. The applications described here

Development, characterization and biocompatibility of chondroitin sulfate/poly(vinyl alcohol)/bovine bone powder porous biocomposite.

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da Silva, Gabriela T; Voss, Guilherme T; Kaplum, Vanessa; Nakamura, Celso V; Wilhelm, Ethel A; Luchese, Cristiane; Fajardo, André R

2017-03-01

Chondroitin sulfate (ChS), a sulfated glycosaminoglycan, poly(vinyl alcohol) (PVA) and bovine bone powder (BBP) were blended to form a novel eco-friendly biocomposite through cyclic freeze-thawing under mild conditions. The systematic investigation reveals that the content of BBP has a remarkable effect on the pore size, porosity, mechanical and liquid uptake properties and biodegradability. At 10wt.% BBP the biocomposite exhibited enhanced mechanical properties and biodegradability rate as compared to the pristine sample. Further, different properties of the biocomposite can be tailored according to the content of BBP. In vitro assays showed that ChS/PVA-BBP does not exert cytotoxicity against healthy cells. In vivo and ex vivo experiments revealed that ChS/PVA-BBP biocomposites are biocompatibility materials without exert pro-inflammatory responses. The biocomposite was completely biodegraded and bioresorbed after 15days of treatment. Taken together, BBP is a low-cost source of hydroxyapatite and collagen, which are insurance. All these results suggest that the biocomposite designed in this study is a promising biomaterial for potential skin tissue engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

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Nilsson, Jonas; Noborn, Fredrik; Gomez Toledo, Alejandro; Nasir, Waqas; Sihlbom, Carina; Larson, Göran

2017-02-01

Purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of glycopeptides, originating from protease digests of glycoproteins, enables site-specific analysis of protein N- and O-glycosylations. We have described a protocol to enrich, hydrolyze by chondroitinase ABC, and characterize chondroitin sulfate-containing glycopeptides (CS-glycopeptides) using positive mode LC-MS/MS. The CS-glycopeptides, originating from the Bikunin proteoglycan of human urine samples, had ΔHexAGalNAcGlcAGalGalXyl- O-Ser hexasaccharide structure and were further substituted with 0-3 sulfate and 0-1 phosphate groups. However, it was not possible to exactly pinpoint sulfate attachment residues, for protonated precursors, due to extensive fragmentation of sulfate groups using high-energy collision induced dissociation (HCD). To circumvent the well-recognized sulfate instability, we now introduced Na+ ions to form sodiated precursors, which protected sulfate groups from decomposition and facilitated the assignment of sulfate modifications. Sulfate groups were pinpointed to both Gal residues and to the GalNAc of the hexasaccharide structure. The intensities of protonated and sodiated saccharide oxonium ions were very prominent in the HCD-MS2 spectra, which provided complementary structural analysis of sulfate substituents of CS-glycopeptides. We have demonstrated a considerable heterogeneity of the bikunin CS linkage region. The realization of these structural variants should be beneficial in studies aimed at investigating the importance of the CS linkage region with regards to the biosynthesis of CS and potential interactions to CS binding proteins. Also, the combined use of protonated and sodiated precursors for positive mode HCD fragmentation analysis will likely become useful for additional classes of sulfated glycopeptides.

Chlorate: a reversible inhibitor of proteoglycan sulfation

International Nuclear Information System (INIS)

Humphries, D.E.; Silbert, J.E.

1988-01-01

Bovine aorta endothelial cells were cultured in medium containing [ 3 H]glucosamine, [ 35 S]sulfate, and various concentrations of chlorate. Cell growth was not affected by 10 mM chlorate, while 30 mM chlorate had a slight inhibitory effect. Chlorate concentrations greater than 10 mM resulted in significant undersulfation of chondroitin. With 30 mM chlorate, sulfation of chondroitin was reduced to 10% and heparan to 35% of controls, but [ 3 H]glucosamine incorporation on a per cell basis did not appear to be inhibited. Removal of chlorate from the culture medium of cells resulted in the rapid resumption of sulfation

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

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Fineran, Paul; Lloyd-Evans, Emyr; Lack, Nathan A; Platt, Nick; Davis, Lianne C; Morgan, Anthony J; Höglinger, Doris; Tatituri, Raju Venkata V; Clark, Simon; Williams, Ian M; Tynan, Patricia; Al Eisa, Nada; Nazarova, Evgeniya; Williams, Ann; Galione, Antony; Ory, Daniel S; Besra, Gurdyal S; Russell, David G; Brenner, Michael B; Sim, Edith; Platt, Frances M

2016-11-18

Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis , achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria

FACE Analysis as a Fast and Reliable Methodology to Monitor the Sulfation and Total Amount of Chondroitin Sulfate in Biological Samples of Clinical Importance

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Evgenia Karousou

2014-06-01

Full Text Available Glycosaminoglycans (GAGs due to their hydrophilic character and high anionic charge densities play important roles in various (pathophysiological processes. The identification and quantification of GAGs in biological samples and tissues could be useful prognostic and diagnostic tools in pathological conditions. Despite the noteworthy progress in the development of sensitive and accurate methodologies for the determination of GAGs, there is a significant lack in methodologies regarding sample preparation and reliable fast analysis methods enabling the simultaneous analysis of several biological samples. In this report, developed protocols for the isolation of GAGs in biological samples were applied to analyze various sulfated chondroitin sulfate- and hyaluronan-derived disaccharides using fluorophore-assisted carbohydrate electrophoresis (FACE. Applications to biologic samples of clinical importance include blood serum, lens capsule tissue and urine. The sample preparation protocol followed by FACE analysis allows quantification with an optimal linearity over the concentration range 1.0–220.0 µg/mL, affording a limit of quantitation of 50 ng of disaccharides. Validation of FACE results was performed by capillary electrophoresis and high performance liquid chromatography techniques.

Quantitative comparison of the efficacy of various compounds in lowering intracellular cholesterol levels in Niemann-Pick type C fibroblasts.

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Zachary T Wehrmann

Full Text Available Niemann-Pick Type C disease (NPC is a lethal, autosomal recessive disorder caused by mutations in the NPC1 and NPC2 cholesterol transport proteins. NPC's hallmark symptoms include an accumulation of unesterified cholesterol and other lipids in the late endosomal and lysosomal cellular compartments, causing progressive neurodegeneration and death. Although the age of onset may vary in those affected, NPC most often manifests in juveniles, and is usually fatal before adolescence. In this study, we investigated the effects of various drugs, many of which modify the epigenetic control of NPC1/NPC2 gene expression, in lowering the otherwise harmful elevated intracellular cholesterol levels in NPC cells. Our studies utilized a previously described image analysis technique, which allowed us to make quantitative comparisons of the efficacy of these drugs in lowering cholesterol levels in a common NPC1 mutant model. Of the drugs analyzed, several that have been previously studied (vorinostat, panobinostat, and β-cyclodextrin significantly lowered the relative amount of unesterified cellular cholesterol, consistent with earlier observations. In addition, a novel potential treatment, rapamycin, likewise alleviated the NPC phenotype. We also studied combinations of effective compounds with β-cyclodextrin; the addition of β-cyclodextrin significantly enhanced the cholesterol-lowering activity of vorinostat and panobinostat, but had mixed effects with rapamycin. Collectively, these results may provide a basis for the eventual development of improved NPC therapies.

Hyaluronic acid and chondroitin sulfate content of osteoarthritic human knee cartilage: site-specific correlation with weight-bearing force based on femorotibial angle measurement.

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Otsuki, Shuhei; Nakajima, Mikio; Lotz, Martin; Kinoshita, Mitsuo

2008-09-01

This study analyzed glycosaminoglycan (GAG) content in specific compartments of the knee joint to determine the impact of malalignment and helped refine indications for osteotomy. To assess malalignment, the radiological femorotibial angle (FTA) was measured and knee joints were also graded for OA severity with the Kellgren/Lawrence (K/L) classification. Cartilage samples were obtained from 36 knees of 32 OA patients undergoing total knee replacement surgery. Explants were harvested from the medial femoral condyle (MFC), lateral femoral condyle (LFC), patellar groove (PG), and lateral posterior femoral condyle (LPC). Concentrations of hyaluronic acid (HA) and chondroitin sulfate (CS) were measured by high-performance liquid chromatography (HPLC). With OA severity, the average FTA significantly increased. HA and CS content in MFC was negatively correlated with radiographic FTA. In LFC, HA ratio, which is HA content in lateral condyle divided by medial condyle and chondroitin 6 sulfate, increased until about 190 degrees FTA. Importantly, at >190 degrees these contents were significantly decreased. HA and CS content of the femoral condyle shows topographic differences that are related to OA grade and weight-bearing force based on FTA. The clinical relevance is that osteotomy may not be indicated for patients with severe varus (>190 degrees) abnormalities. (c) 2008 Orthopaedic Research Society

Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C.

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Lopez, Manuel E; Klein, Andrés D; Hong, Jennifer; Dimbil, Ubah J; Scott, Matthew P

2012-07-01

Chronic systemic inflammation is thought to be a major contributor to metabolic and neurodegenerative diseases. Since inflammatory components are shared among different disorders, targeting inflammation is an attractive option for mitigating disease. To test the significance of inflammation in the lipid storage disorder (LSD) Niemann-Pick C (NPC), we deleted the macrophage inflammatory gene Mip1a/Ccl3 from NPC diseased mice. Deletion of Ccl3 had been reported to delay neuronal loss in Sandhoff LSD mice by inhibiting macrophage infiltration. For NPC mice, in contrast, deleting Ccl3 did not retard neurodegeneration and worsened the clinical outcome. Depletion of visceral tissue macrophages also did not alter central nervous system (CNS) pathology and instead increased liver injury, suggesting a limited macrophage infiltration response into the CNS and a beneficial role of macrophage activity in visceral tissue. Prevention of neuron loss or liver injury, even at late stages in the disease, was achieved through specific rescue of NPC disease in neurons or in liver epithelial cells, respectively. Local epithelial cell correction was also sufficient to reduce the macrophage-associated pathology in lung tissue. These results demonstrate that elevated inflammation and macrophage activity does not necessarily contribute to neurodegeneration and tissue injury, and LSD defects in immune cells may not preclude an appropriate inflammatory response. We conclude that inflammation remains secondary to neuronal and epithelial cell dysfunction and does not irreversibly contribute to the pathogenic cascade in NPC disease. Without further exploration of possible beneficial roles of inflammatory mediators, targeting inflammation may not be therapeutically effective at ameliorating disease severity.

Genetic and Chemical Correction of Cholesterol Accumulation and Impaired Autophagy in Hepatic and Neural Cells Derived from Niemann-Pick Type C Patient-Specific iPS Cells

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Dorothea Maetzel

2014-06-01

Full Text Available Niemann-Pick type C (NPC disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs using transcription activator-like effector nucleases (TALENs. We observed decreased cell viability, cholesterol accumulation, and dysfunctional autophagic flux in NPC1-deficient human hepatic and neural cells. Genetic correction of a disease-causing mutation rescued these defects and directly linked NPC1 protein function to impaired cholesterol metabolism and autophagy. Screening for autophagy-inducing compounds in disease-affected human cells showed cell type specificity. Carbamazepine was found to be cytoprotective and effective in restoring the autophagy defects in both NPC1-deficient hepatic and neuronal cells and therefore may be a promising treatment option with overall benefit for NPC disease.

Plasma antibodies from malaria-exposed pregnant women recognize variant surface antigens on Plasmodium falciparum-infected erythrocytes in a parity-dependent manner and block parasite adhesion to chondroitin sulfate A

DEFF Research Database (Denmark)

Ricke, C H; Staalsoe, T; Koram, K

2000-01-01

-associated malaria (PAM) in endemic areas is concentrated in the first few pregnancies, indicating that protective immunity to PAM is a function of parity. The placenta is often heavily infected in PAM, and placental parasites show a striking preference for chondroitin sulfate A (CSA) as an adhesion receptor. Plasma...

Plasma lysosphingomyelin demonstrates great potential as a diagnostic biomarker for Niemann-Pick disease type C in a retrospective study.

Directory of Open Access Journals (Sweden)

Richard W D Welford

Full Text Available Niemann-Pick disease type C (NP-C is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC and glucosylsphingosine (GlcSph, appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naïve NP-C patients, aged 2-50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.

Synthesis, characterization, and evaluation of pluronic-based β-cyclodextrin polyrotaxanes for mobilization of accumulated cholesterol from Niemann-Pick type C fibroblasts.

Science.gov (United States)

Collins, Christopher J; McCauliff, Leslie A; Hyun, Seok-Hee; Zhang, Zhaorui; Paul, Lake N; Kulkarni, Aditya; Zick, Klaus; Wirth, Mary; Storch, Judith; Thompson, David H

2013-05-14

Several lines of evidence suggest that β-cyclodextrin (β-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current β-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic surfactant-based β-CD polyrotaxanes has been developed using biocompatible poly(ethylene glycol) (PEG)-polypropylene glycol (PPG)-PEG triblock copolymers. These compounds carry multiple copies of β-CD as shown by (1)H NMR, 2D nuclear Overhouser effect spectroscopy, gel permeation chromatography/multiangle light scattering, analytical ultracentrifugation analysis, matrix assisted laser desorption/ionization mass spectrometry, and diffusion-ordered spectroscopy. Analyses of free β-cyclodextrin contamination in the compounds were made by reverse phase high pressure liquid chromatography and hydrophilic interaction liquid chromatography. Dethreading kinetics were studied by reverse phase high pressure liquid chromatography, UV/vis, and (1)H NMR analysis. Filipin staining studies using npc2(-/-) fibroblasts show significant reversal of cholesterol accumulation after treatment with polyrotaxane compounds. The rate and efficacy of reversal is similar to that achieved by equivalent amounts of monomeric β-CD alone.

Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone.

Science.gov (United States)

Gui, Meng; Song, Juyi; Zhang, Lu; Wang, Shun; Wu, Ruiyun; Ma, Changwei; Li, Pinglan

2015-06-05

Chondroitin sulfates (CSs) were extracted from sturgeon skull and backbone, and their chemical composition, anticoagulant, anti-platelet and thrombolysis activities were evaluated. The average molecular weights of CS from sturgeon skull and backbone were 38.5kDa and 49.2kDa, respectively. Disaccharide analysis indicated that the sturgeon backbone CS was primarily composed of disaccharide monosulfated in position four of the GalNAc (37.8%) and disaccharide monosulfated in position six of the GalNAc (59.6%) while sturgeon skull CS was primarily composed of nonsulfated disaccharide (74.2%). Sturgeon backbone CS showed stronger antithrombotic effect than sturgeon skull CS. Sturgeon backbone CS could significantly prolong activated partial thromboplastin time (APTT) and thrombin time (TT), inhibited ADP-induced platelet aggregation and dissolved platelet plasma clots in vitro. The results suggested that sturgeon backbone CS can be explored as a functional food with antithrombotic function. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preparation and optimization of self-assembled chondroitin sulfate-nisin nanogel based on quality by design concept.

Science.gov (United States)

Mohtashamian, Shahab; Boddohi, Soheil; Hosseinkhani, Saman

2018-02-01

Self-assembled nanogel was prepared by electrostatic complexation of two oppositely charged biological macromolecules, which were cationic nisin and anionic chondroitin sulfate (ChS). The critical factors affected the physical properties of ChS-nisin nanogel was screened and optimized by Plackett-Burman design (PB) and central composite design (CCD). The independent factors selected were: concentration ratio of nisin to ChS, injection rate of nisin solution, buffer solvent type, magnetic stirring rate, pH of initial buffer solution, centrifuge-cooling temperature, and centrifuge rotation speed. Among these factors, concentration ratio changed the entrapment efficiency and loading capacity significantly. In addition, the hydrodynamic diameter and loading capacity were significantly influenced by injection rate and pH of initial buffer solution. The optimized nanogel structure was obtained by concentration ratio of 6.4mg/mL nisin to 1mg/mL ChS, pH of buffer solution at 4.6, and nisin solution injection rate of 0.2mL/min. The observed values of dependent responses were close to predicted values confirmed by model from response surface methodology. The results obviously showed that quality by design concept (QbD) could be effectively applied to optimize the developed ChS-nisin nanogel. Copyright © 2017 Elsevier B.V. All rights reserved.

Preparation and characterization of chondroitin-sulfate-A-coated magnetite nanoparticles for biomedical applications

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Tóth, Ildikó Y.; Illés, Erzsébet; Szekeres, Márta; Tombácz, Etelka

2015-04-01

Polysaccharides are promising candidates for manufacturing biocompatible core-shell nanoparticles with potential in vivo use. Superparamagnetic magnetite nanoparticles (MNPs) have prospective application in both diagnosis and therapy, and so developing a novel polysaccharide shell on MNP core is of great challenge. MNPs were prepared by co-precipitation, then the surface of purified MNPs was coated with chondroitin-sulfate-A (CSA) to obtain core-shell structured magnetite nanoparticles (CSA@MNP). The effect of the added amount of CSA on the surface charging and the aggregation state of MNPs at various pHs and 10 mM NaCl was measured by electrophoresis and dynamic light scattering. The amphoteric behavior of MNPs was fundamentally modified by adsorption of CSA polyanions. A very low CSA-loading induces the aggregation of MNPs, while four times more stabilizes the dispersions over the whole pH-range studied. The coagulation kinetics experiments measured at pH=6.3±0.3 showed that salt tolerance of CSA@MNPs rises up to ~150 mM NaCl.

Multiple cationic amphiphiles induce a Niemann-Pick C phenotype and inhibit Ebola virus entry and infection.

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Charles J Shoemaker

Full Text Available Ebola virus (EBOV is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC(50 1.6 to 8.0 µM at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann-Pick C1 protein (NPC1, a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.

Psychiatric and neurological symptoms in patients with Niemann-Pick disease type C (NP-C): Findings from the International NPC Registry.

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Bonnot, Olivier; Gama, Clarissa S; Mengel, Eugen; Pineda, Mercè; Vanier, Marie T; Watson, Louise; Watissée, Marie; Schwierin, Barbara; Patterson, Marc C

2017-10-09

Niemann-Pick disease type C (NP-C) is a rare inherited neurovisceral disease that should be recognised by psychiatrists as a possible underlying cause of psychiatric abnormalities. This study describes NP-C patients who had psychiatric manifestations at enrolment in the international NPC Registry, a unique multicentre, prospective, observational disease registry. Treating physicians' data entries describing psychiatric manifestations in NPC patients were coded and grouped by expert psychiatrists. Out of 386 NP-C patients included in the registry as of October 2015, psychiatric abnormalities were reported to be present in 34% (94/280) of those with available data. Forty-four patients were confirmed to have identifiable psychiatric manifestations, with text describing these psychiatric manifestations. In these 44 patients, the median (range) age at onset of psychiatric manifestations was 17.9 years (2.5-67.9; n = 15), while the median (range) age at NP-C diagnosis was 23.7 years (0.2-69.8; n = 34). Almost all patients (43/44; 98%) had an occurrence of ≥1 neurological manifestation at enrolment. These data show that substantial delays in diagnosis of NP-C are long among patients with psychiatric symptoms and, moreover, patients presenting with psychiatric features and at least one of cognitive impairment, neurological manifestations, and/or visceral symptoms should be screened for NP-C.

Disruption in connexin-based communication is associated with intracellular Ca²⁺ signal alterations in astrocytes from Niemann-Pick type C mice.

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Pablo J Sáez

Full Text Available Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻ showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

Disruption in connexin-based communication is associated with intracellular Ca²⁺ signal alterations in astrocytes from Niemann-Pick type C mice.

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Sáez, Pablo J; Orellana, Juan A; Vega-Riveros, Natalia; Figueroa, Vania A; Hernández, Diego E; Castro, Juan F; Klein, Andrés D; Jiang, Jean X; Zanlungo, Silvana; Sáez, Juan C

2013-01-01

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

Collaborative Development of 2-Hydroxypropyl-β-Cyclodextrin for the Treatment of Niemann-Pick Type C1 Disease

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Ottinger, Elizabeth A.; Kao, Mark L.; Carrillo-Carrasco, Nuria; Yanjanin, Nicole; Shankar, Roopa Kanakatti; Janssen, Marjo; Brewster, Marcus; Scott, Ilona; Xu, Xin; Cradock, Jim; Terse, Pramod; Dehdashti, Seameen; Marugan, Juan; Zheng, Wei; Portilla, Lili; Hubbs, Alan; Pavan, William J.; Heiss, John; Vite, Charles H.; Walkley, Steven U.; Ory, Daniel S.; Silber, Steven A.; Porter, Forbes D.; Austin, Christopher P.; McKew, John C.

2014-01-01

In 2010, the National Institutes of Health (NIH) established the Therapeutics for Rare and Neglected Diseases (TRND) program within the National Center for Advancing Translational Science (NCATS), which was created to stimulate drug discovery and development for rare and neglected tropical diseases through a collaborative model between the NIH, academic scientists, nonprofit organizations, and pharmaceutical and biotechnology companies. This paper describes one of the first TRND programs, the development of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) for the treatment of Niemann-Pick disease type C1 (NPC1). NPC is a neurodegenerative, autosomal recessive rare disease caused by a mutation in either the NPC1 (about 95% of cases) or the NPC2 gene (about 5% of cases). These mutations affect the intracellular trafficking of cholesterol and other lipids, which leads to a progressive accumulation of unesterified cholesterol and glycosphingolipids in the CNS and visceral organs. Affected individuals typically exhibit ataxia, swallowing problems, seizures, and progressive impairment of motor and intellectual function in early childhood, and usually die in adolescence. There is no disease modifying therapy currently approved for NPC1 in the US. A collaborative drug development program has been established between TRND, public and private partners that has completed the pre-clinical development of HP-β-CD through IND filing for the current Phase I clinical trial that is underway. Here we discuss how this collaborative effort helped to overcome scientific, clinical and financial challenges facing the development of new drug treatments for rare and neglected diseases, and how it will incentivize the commercialization of HP-β-CD for the benefit of the NPC patient community. PMID:24283970

Cyclodextrin alleviates neuronal storage of cholesterol in Niemann-Pick C disease without evidence of detectable blood-brain barrier permeability.

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Pontikis, Charles C; Davidson, Cristin D; Walkley, Steven U; Platt, Frances M; Begley, David J

2013-05-01

Niemann-Pick type C disease is an inherited autosomal recessive neurodegenerative disorder characterised by the accumulation of unesterified cholesterol and sphingolipids within the endosomal/lysosomal compartments. It has been observed that the administration of hydroxypropyl-β-cyclodextrin (HPBCD) delays onset of clinical symptoms and reduces accumulation of cholesterol and gangliosides within neuronal cells. It was assumed that HPBCD exerts its action by readily entering the CNS and directly interacting with neurones and other brain cells to facilitate removal of stored cholesterol from the late endosomal/lysosomal compartment. Here, we present evidence that refutes this hypothesis. We use two well established techniques for accurately measuring brain uptake of solutes from blood and show that there is no significant crossing of HPBCD into the brain. The two techniques are brain in situ perfusion and intraperitoneal injection followed by multi-time-point regression analysis. Neither study demonstrates significant, time-dependent uptake of HPBCD in either adult or neonatal mice. However, the volume of distribution available to HPBCD (0.113 ± 0.010 ml/g) exceeds the accepted values for plasma and vascular volume of the brain. In fact, it is nearly three times larger than that for sucrose (0.039 ± 0.006 ml/g). We propose that this indicates cell surface binding of HPBCD to the endothelium of the cerebral vasculature and may provide a mechanism for the mobilisation and clearance of cholesterol from the CNS.

Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

International Nuclear Information System (INIS)

Kosicek, Marko; Malnar, Martina; Goate, Alison; Hecimovic, Silva

2010-01-01

It has been suggested that cholesterol may modulate amyloid-β (Aβ) formation, a causative factor of Alzheimer's disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (β-amyloid precursor protein (APP), β-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Aβ formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1 -/- cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, γ-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Aβ occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer's disease and supports the role of lipid rafts in these processes.

Pharmacologic Treatment Assigned for Niemann Pick Type C1 Disease Partly Changes Behavioral Traits in Wild-Type Mice.

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Schlegel, Victoria; Thieme, Markus; Holzmann, Carsten; Witt, Martin; Grittner, Ulrike; Rolfs, Arndt; Wree, Andreas

2016-11-09

Niemann-Pick Type C1 (NPC1) is an autosomal recessive inherited disorder characterized by accumulation of cholesterol and glycosphingolipids. Previously, we demonstrated that BALB/c-npc1 nih Npc1 -/- mice treated with miglustat, cyclodextrin and allopregnanolone generally performed better than untreated Npc1 -/- animals. Unexpectedly, they also seemed to accomplish motor tests better than their sham-treated wild-type littermates. However, combination-treated mutant mice displayed worse cognition performance compared to sham-treated ones. To evaluate effects of these drugs in healthy BALB/c mice, we here analyzed pharmacologic effects on motor and cognitive behavior of wild-type mice. For combination treatment mice were injected with allopregnanolone/cyclodextrin weekly, starting at P7. Miglustat injections were performed daily from P10 till P23. Starting at P23, miglustat was embedded in the chow. Other mice were treated with miglustat only, or sham-treated. The battery of behavioral tests consisted of accelerod, Morris water maze, elevated plus maze, open field and hot-plate tests. Motor capabilities and spontaneous motor behavior were unaltered in both drug-treated groups. Miglustat-treated wild-type mice displayed impaired spatial learning compared to sham- and combination-treated mice. Both combination- and miglustat-treated mice showed enhanced anxiety in the elevated plus maze compared to sham-treated mice. Additionally, combination treatment as well as miglustat alone significantly reduced brain weight, whereas only combination treatment reduced body weight significantly. Our results suggest that allopregnanolone/cyclodextrin ameliorate most side effects of miglustat in wild-type mice.

Pharmacologic Treatment Assigned for Niemann Pick Type C1 Disease Partly Changes Behavioral Traits in Wild-Type Mice

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Victoria Schlegel

2016-11-01

Full Text Available Niemann-Pick Type C1 (NPC1 is an autosomal recessive inherited disorder characterized by accumulation of cholesterol and glycosphingolipids. Previously, we demonstrated that BALB/c-npc1nihNpc1−/− mice treated with miglustat, cyclodextrin and allopregnanolone generally performed better than untreated Npc1−/− animals. Unexpectedly, they also seemed to accomplish motor tests better than their sham-treated wild-type littermates. However, combination-treated mutant mice displayed worse cognition performance compared to sham-treated ones. To evaluate effects of these drugs in healthy BALB/c mice, we here analyzed pharmacologic effects on motor and cognitive behavior of wild-type mice. For combination treatment mice were injected with allopregnanolone/cyclodextrin weekly, starting at P7. Miglustat injections were performed daily from P10 till P23. Starting at P23, miglustat was embedded in the chow. Other mice were treated with miglustat only, or sham-treated. The battery of behavioral tests consisted of accelerod, Morris water maze, elevated plus maze, open field and hot-plate tests. Motor capabilities and spontaneous motor behavior were unaltered in both drug-treated groups. Miglustat-treated wild-type mice displayed impaired spatial learning compared to sham- and combination-treated mice. Both combination- and miglustat-treated mice showed enhanced anxiety in the elevated plus maze compared to sham-treated mice. Additionally, combination treatment as well as miglustat alone significantly reduced brain weight, whereas only combination treatment reduced body weight significantly. Our results suggest that allopregnanolone/cyclodextrin ameliorate most side effects of miglustat in wild-type mice.

Silk fibroin/gelatin-chondroitin sulfate-hyaluronic acid effectively enhances in vitro chondrogenesis of bone marrow mesenchymal stem cells.

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Sawatjui, Nopporn; Damrongrungruang, Teerasak; Leeanansaksiri, Wilairat; Jearanaikoon, Patcharee; Hongeng, Suradej; Limpaiboon, Temduang

2015-01-01

Tissue engineering is becoming promising for cartilage repair due to the limited self-repair capacity of cartilage tissue. We previously fabricated and characterized a three-dimensional silk fibroin/gelatin-chondroitin sulfate-hyaluronic acid (SF-GCH) scaffold and showed that it could promote proliferation of human bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate its biological performance as a new biomimetic material for chondrogenic induction of BM-MSCs in comparison to an SF scaffold and conventional pellet culture. We found that the SF-GCH scaffold significantly enhanced the proliferation and chondrogenic differentiation of BM-MSCs compared to the SF scaffold and pellet culture in which the production of sulfated glycoaminoglycan was increased in concordance with the up-regulation of chondrogenic-specific gene markers. Our findings indicate the significant role of SF-GCH by providing a supportive structure and the mimetic cartilage environment for chondrogenesis which enables cartilage regeneration. Thus, our fabricated SF-GCH scaffold may serve as a potential biomimetic material for cartilage tissue engineering. Copyright © 2015 Elsevier B.V. All rights reserved.

Defining natural history: assessment of the ability of college students to aid in characterizing clinical progression of Niemann-Pick disease, type C.

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Jenny Shin

Full Text Available Niemann-Pick Disease, type C (NPC is a fatal, neurodegenerative, lysosomal storage disorder. It is a rare disease with broad phenotypic spectrum and variable age of onset. These issues make it difficult to develop a universally accepted clinical outcome measure to assess urgently needed therapies. To this end, clinical investigators have defined emerging, disease severity scales. The average time from initial symptom to diagnosis is approximately 4 years. Further, some patients may not travel to specialized clinical centers even after diagnosis. We were therefore interested in investigating whether appropriately trained, community-based assessment of patient records could assist in defining disease progression using clinical severity scores. In this study we evolved a secure, step wise process to show that pre-existing medical records may be correctly assessed by non-clinical practitioners trained to quantify disease progression. Sixty-four undergraduate students at the University of Notre Dame were expertly trained in clinical disease assessment and recognition of major and minor symptoms of NPC. Seven clinical records, randomly selected from a total of thirty seven used to establish a leading clinical severity scale, were correctly assessed to show expected characteristics of linear disease progression. Student assessment of two new records donated by NPC families to our study also revealed linear progression of disease, but both showed accelerated disease progression, relative to the current severity scale, especially at the later stages. Together, these data suggest that college students may be trained in assessment of patient records, and thus provide insight into the natural history of a disease.

Brain ageing changes proteoglycan sulfation, rendering perineuronal nets more inhibitory.

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Foscarin, Simona; Raha-Chowdhury, Ruma; Fawcett, James W; Kwok, Jessica C F

2017-06-28

Chondroitin sulfate (CS) proteoglycans in perineuronal nets (PNNs) from the central nervous system (CNS) are involved in the control of plasticity and memory. Removing PNNs reactivates plasticity and restores memory in models of Alzheimer's disease and ageing. Their actions depend on the glycosaminoglycan (GAG) chains of CS proteoglycans, which are mainly sulfated in the 4 (C4S) or 6 (C6S) positions. While C4S is inhibitory, C6S is more permissive to axon growth, regeneration and plasticity. C6S decreases during critical period closure. We asked whether there is a late change in CS-GAG sulfation associated with memory loss in aged rats. Immunohistochemistry revealed a progressive increase in C4S and decrease in C6S from 3 to 18 months. GAGs extracted from brain PNNs showed a large reduction in C6S at 12 and 18 months, increasing the C4S/C6S ratio. There was no significant change in mRNA levels of the chondroitin sulfotransferases. PNN GAGs were more inhibitory to axon growth than those from the diffuse extracellular matrix. The 18-month PNN GAGs were more inhibitory than 3-month PNN GAGs. We suggest that the change in PNN GAG sulfation in aged brains renders the PNNs more inhibitory, which lead to a decrease in plasticity and adversely affect memory.

Treatment of Chronic Skin-Picking in an Adolescent With Asperger Syndrome and Borderline Intellectual Disability

NARCIS (Netherlands)

Lang, R.B.; Didden, H.C.M.; Sigafoos, J.; Rispoli, M.; Regester, A.; Lancioni, G.E.

2009-01-01

We present the case of a 17-year-old girl with Asperger syndrome and borderline intellectual disability with a 5-year history of chronic skin-picking. Our intervention approach included an initial functional assessment to identify variables maintaining skin-picking, followed by evaluation of a

Effects of retinal growth factor and of the increase of the number of subcultures on sulfated glycosaminoglycans of bovine lens epithelial cells

International Nuclear Information System (INIS)

Moczar, E.; Courtois, Y.

1981-01-01

Sulfated glycosaminoglycans of cultured bovine lens epithelial cells grown in the presence and in the absence of a retinal growth factor were investigated comparatively. The newly formed [ 35 S] sulfate-labeled glycosaminoglycans were analysed in the extra-, peri- and intracellular compartments of early (4-5th) and late (17-18h) subcultures. The following results were obtained: (1) Cultured lens epithelial cells grown in the presence or in the absence of the growth factor synthesize chondroitin 4- and 6-sulfates and dermatan sulfate, with heparan sulfate as the main component, the pericellular compartments were particularly rich in heparan sulfate; (2) The distribution pattern of the glycosaminoglycans changes during successive subcultures; the proportion of heparan sulfate increases in the pericellular compartment, the dermatan sulfate to chondroitin sulfate ratio increases in all three compartments; (3) In contrast to the drastic decrease in the fibronectin levels in the presence of growth factor in the early subcultures, only minor differences were found between the glycosaminoglycan patterns for the treated and non-treated cells. ( orig.)

Critical appraisal of the role of glucosamine and chondroitin in the management of osteoarthritis of the knee

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Steven J Narvy

2010-02-01

Full Text Available Steven J Narvy1, C Thomas Vangsness Jr21Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 2Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USAAbstract: Osteoarthritis (OA is the most common musculoskeletal disease in the United States, with rising prevalence. Medical management of OA involves acetaminophen, nonsteroidal anti-inflammatory drugs, and other analgesics, all of which are of variable efficacy and are associated with significant side effects and toxicities. The purpose of this review is to critically evaluate the efficacy of glucosamine and chondroitin, both as single agents and in combination, for the treatment of knee OA. Also evaluated were the level of evidence and funding support of the included articles. Almost every included trial of glucosamine sulfate, glucosamine hydrochloride, and chondroitin sulfate has found the safety of these compounds to be equal to that of placebo, though their therapeutic efficacy in decreasing knee OA pain and improving joint function is variable. Additionally, there are data to support a role of these agents in reducing radiographic progression of knee OA. Industry involvement, however, remains prominent. Further, more comprehensive study by independent researchers free of industry ties is necessary to identify a subset of patients in whom the use of glucosamine and/or chondroitin would be most beneficial. These agents may be safely tried as an initial therapy in select OA patients prior to initiating therapy with nonsteroidal anti-inflammatory drugs, acetaminophen, and other traditional medications.Keywords: glucosamine sulfate, glucosamine hydrochloride, chondroitin sulfate, knee osteoarthritis, nutritional supplement, nutraceutical

Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1

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Mao, Yang; Resende, Mafalda; Daugaard, Mads; Riis Kristensen, Anders; Damm, Peter; G. Theander, Thor; R. Hansson, Stefan; Salanti, Ali

2016-01-01

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells. PMID:27556547

Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor

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Sainz, Bruno; Barretto, Naina; Martin, Danyelle N.; Hiraga, Nobuhiko; Imamura, Michio; Hussain, Snawar; Marsh, Katherine A.; Yu, Xuemei; Chayama, Kazuaki; Alrefai, Waddah A.; Uprichard, Susan L.

2011-01-01

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ~170 million individuals infected and current interferon-based treatment having toxic side-effects and marginal efficacy, more effective antivirals are critically needed1. Although HCV protease inhibitors were just FDA approved, analogous to HIV therapy, optimal HCV therapy likely will require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a promising multi-faceted target for antiviral intervention; however, to date FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1-Like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection as silencing or antibody-mediated blocking of NPC1L1 impairs cell-cultured-derived HCV (HCVcc) infection initiation. In addition, the clinically-available FDA-approved NPC1L1 antagonist ezetimibe2,3 potently blocks HCV uptake in vitro via a virion cholesterol-dependent step prior to virion-cell membrane fusion. Importantly, ezetimibe inhibits infection of all major HCV genotypes in vitro, and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor, but also discovered a new antiviral target and potential therapeutic agent. PMID:22231557

Evaluation of an anti-tumor necrosis factor therapeutic in a mouse model of Niemann-Pick C liver disease.

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Melanie Vincent

2010-09-01

Full Text Available Niemann-Pick type C (NPC disease is a lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. The majority of NPC patients die in their teen years due to progressive neurodegeneration; however, half of NPC patients also suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. We previously showed that a key mediator of NPC liver disease is tumor necrosis factor (TNF α, which is involved in both proinflammatory and apoptotic signaling cascades. In this study, we tested the hypothesis that blocking TNF action with an anti-TNF monoclonal antibody (CNTO5048 will slow the progression of NPC liver disease.Treatment of wild-type C57BL/6 mice with NPC1-specific antisense oligonucleotides led to knockdown of NPC1 protein expression in the liver. This caused classical symptoms of NPC liver disease, including hepatic cholesterol accumulation, hepatomegaly, elevated serum liver enzymes, and lipid laden macrophage accumulation. In addition, there was a significant increase in the number of apoptotic cells and a proliferation of stellate cells. Concurrent treatment of NPC1 knockdown mice with anti-TNF had no effect on the primary lipid storage or accumulation of lipid-laden macrophages. However, anti-TNF treatment slightly blunted the increase in hepatic apoptosis and stellate cell activation that was seen with NPC1 knockdown.Current therapeutic options for NPC disease are limited. Our results provide proof of principle that pharmacologically blocking the TNF-α inflammatory cascade can slightly reduce certain markers of NPC disease. Small molecule inhibitors of TNF that penetrate tissues and cross the blood-brain barrier may prove even more beneficial.

Functional Assessment and Behavioral Treatment of Skin Picking in a Teenage Girl With Prader-Willi Syndrome

NARCIS (Netherlands)

Radstaake, M.; Didden, H.C.M.; Bolio, M.M.L.; Lang, R.B.; Lancioni, G.E.; Curfs, L.M.G.

2011-01-01

Skin picking is common in individuals with Prader-Willi Syndrome (PWS) but few treatment studies exist. This study reports the successful functional behavioral assessment (FBA) and treatment of skin picking in a 16-year-old female with PWS. A treatment package based on FBA results consisted of

Chondroitin sulfate reduces the friction coefficient of articular cartilage.

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Basalo, Ines M; Chahine, Nadeen O; Kaplun, Michael; Chen, Faye H; Hung, Clark T; Ateshian, Gerard A

2007-01-01

The objective of this study was to investigate the effect of chondroitin sulfate (CS)-C on the frictional response of bovine articular cartilage. The main hypothesis is that CS decreases the friction coefficient of articular cartilage. Corollary hypotheses are that viscosity and osmotic pressure are not the mechanisms that mediate the reduction in the friction coefficient by CS. In Experiment 1, bovine articular cartilage samples (n=29) were tested in either phosphate buffered saline (PBS) or in PBS containing 100mg/ml of CS following 48h incubation in PBS or in PBS+100mg/ml CS (control specimens were not subjected to any incubation). In Experiment 2, samples (n=23) were tested in four different solutions: PBS, PBS+100mg/ml CS, and PBS+polyethylene glycol (PEG) (133 or 170mg/ml). In Experiment 3, samples (n=18) were tested in three solutions of CS (0, 10 and 100mg/ml). Frictional tests (cartilage-on-glass) were performed under constant stress (0.5MPa) for 3600s and the time-dependent friction coefficient was measured. Samples incubated or tested in a 100mg/ml CS solution exhibited a significantly lower equilibrium friction coefficient than the respective PBS control. PEG solutions delayed the rise in the friction coefficient relative to the PBS control, but did not reduce the equilibrium value. Testing in PBS+10mg/ml of CS did not cause any significant decrease in the friction coefficient. In conclusion, CS at a concentration of 100mg/ml significantly reduces the friction coefficient of bovine articular cartilage and this mechanism is neither mediated by viscosity nor osmolarity. These results suggest that direct injection of CS into the joint may provide beneficial tribological effects.

Chondroitin for osteoarthritis

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Singh, Jasvinder A.; Noorbaloochi, Shahrzad; MacDonald, Roderick; Maxwell, Lara J.

2016-01-01

Background Osteoarthritis, a common joint disorder, is one of the leading causes of disability. Chondroitin has emerged as a new treatment. Previous meta-analyses have shown contradictory results on the efficacy of chondroitin. This, in addition to the publication of more trials, necessitates a systematic review. Objectives To evaluate the benefit and harm of oral chondroitin for treating osteoarthritis compared with placebo or a comparator oral medication including, but not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, opioids, and glucosamine or other “herbal” medications. Search methods We searched seven databases up to November 2013, including the Cochrane Central Register of Controlled Trials (CENTRAL), Ovid MEDLINE, CINAHL, EMBASE, Science Citation Index (Web of Science) and Current Controlled Trials. We searched the US Food and Drug Administration (FDA) and European Medicines Agency (EMEA) websites for adverse effects. Trial registers were not searched. Selection criteria All randomized or quasi-randomized clinical trials lasting longer than two weeks, studying adults with osteoarthritis in any joint, and comparing chondroitin with placebo, an active control such as NSAIDs, or other “herbal” supplements such as glucosamine. Data collection and analysis Two review authors independently performed all title assessments, data extractions, and risk of bias assessments. Main results Forty-three randomized controlled trials including 4,962 participants treated with chondroitin and 4,148 participants given placebo or another control were included. The majority of trials were in knee OA, with few in hip and hand OA. Trial duration varied from 1 month to 3 years. Participants treated with chondroitin achieved statistically significantly and clinically meaningful better pain scores (0–100) in studies less than 6 months than those given placebo with an absolute risk difference of 10% lower (95% confidence interval (CI), 15% to 6% lower

Glucosamine and chondroitin use in canines for osteoarthritis: A review

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Angel Bhathal

2017-02-01

Full Text Available Osteoarthritis is a slowly progressive and debilitating disease that affects canines of all breeds. Pain and decreased mobility resulting from osteoarthritis often have a negative impact on the affected canine’s quality of life, level of comfort, daily functioning, activity, behaviour, and client-pet companionship. Despite limited and conflicting evidence, the natural products glucosamine hydrochloride (HCl and chondroitin sulfate are commonly recommended by veterinarians for treating osteoarthritis in dogs. There is a paucity of well-designed clinical veterinary studies investigating the true treatment effect of glucosamine and chondroitin. The purposes of this review article are to provide a brief background on glucosamine and chondroitin use in canine osteoarthritis and to critically review the available literature on the role of these products for improving clinical outcomes. Based on critical review, recommendations for practice are suggested and a future study design is proposed.

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease.

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Chan Chung

2016-05-01

Full Text Available Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1, promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.

Fucosylated chondroitin sulfate is covalently associated with collagen fibrils in sea cucumber Apostichopus japonicus body wall.

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Wang, Jun; Chang, Yaoguang; Wu, Fanxiu; Xu, Xiaoqi; Xue, Changhu

2018-04-15

Fucosylated chondroitin sulfate (fCS) is the major carbohydrate constituent of sea cucumber. However, the distribution of fCS in the sea cucumber body wall has not been fully described. We addressed this in the present study employing Apostichopus japonicus as the material, a sea cucumber species with significant commercial importance. It was found that fCS was covalently attached to collagen fibrils via O-glycosidic linkages. Transmission electron microscopy analysis revealed that fCS precipitate was present in gap regions of collagen fibrils as roughly globular or ellipsoidal dots. The fCS dots arranged circumferentially around the fibrils with an axial repeat period that matched the periodicity of the fibrils. Physicochemical analysis indicated that the presence of fCS significantly increased the negative charge of the fibrils. These findings provide novel insight into fCS distribution in the sea cucumber body wall and its supramolecular organization with other macromolecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

Chondroitin sulfate iron colloid as MR contrast agent in differentiation between hepatocellular carcinoma and adenomatous hyperplasia

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Suto, Y. (Department of Radiology, Tottori Univ. School of Medicine, Yonago (Japan)); Kato, T. (Department of Radiology, Tottori Univ. School of Medicine, Yonago (Japan)); Matsuo, T. (Department of Radiology, Tottori Univ. School of Medicine, Yonago (Japan)); Kamba, M. (Department of Radiology, Tottori Univ. School of Medicine, Yonago (Japan)); Shimatani, Y. (Department of Radiology, Tottori Univ. School of Medicine, Yonago (Japan)); Ohuchi, Y. (Department of Radiology, Tottori Univ. School of Medicine, Yonago (Japan)); Nakamura, K. (Department of Radiology, Tottori Univ. School of Medicine, Yonago (Japan)); Ohta, Y. (Department of Radiology, Tottori Univ. School of Medicine, Yonago (Japan))

1993-05-01

Using a 1.5 T MR imaging unit, T1- and T2-weighted images were obtained before and after i.v. administration of chondroitin sulfate iron colloid (CSIC) in order to differentiate hepatocellular carcinoma (n=20) from adenomatous hyperplasia without atypia (n=16). Differentiation was made from the tumor-liver contrast to noise ratio (CNR) and visual evaluation of the nodule, with reference to signal intensity relative to that of the surrounding liver. The CNR of adenomatous hyperplasia was on T1-weighted images significantly decreased after CSIC administration (p<0.01). On T2-weighted images, there was no significant difference in CNR after CSIC administration. On the other hand, the CNR of hepatocellular carcinoma was significantly increased after CSIC administration on both T1- and T2-weighted images (p<0.01). CSIC reflects intratumor reticuloendothelial cellular functions, and is therefore useful in differentiating hepatocellular carcinoma from adenomatous hyperplasia without atypia. (orig.).

Chondroitin sulfate and glucosamine sulfate associated to photobiomodulation prevents degenerative morphological changes in an experimental model of osteoarthritis in rats.

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Sanches, Marcella; Assis, Lívia; Criniti, Cyntia; Fernandes, Danilo; Tim, Carla; Renno, Ana Claudia Muniz

2018-04-01

The aim of this study was to compare the effects of combined treatment with chondroitin sulfate and glucosamine sulfate (CS/Gl) and photobiomodulation (PBM) on the degenerative process related to osteoarthritis (OA) in the articular cartilage in rats. Forty male Wistar rats were randomly divided into four groups: OA control group (CG); OA animals submitted to PBM treatment (PBM); OA animals submitted to CS/Gl treatment (CS/Gl); OA submitted to CS/GS associated with PBM treatments (GS/Gl + PBM). The CS/Gl started 48 h after the surgery, and they were performed for 29 consecutive days. Moreover, PBM was performed after the CS/Gl administration on the left joint. Morphological characteristics and immunoexpression of interleukin 10 (IL-10) and 1 beta (IL-1β) and collagen type II (Col II) of the articular cartilage were evaluated. The results showed that all treated groups (CS/Gl and PBM) presented attenuation signs of degenerative process (measured by histopathological analysis) and lower density chondrocytes [PBM (p = 0.0017); CS/Gl (p = 0.0153) and CS/Gl + PBM (p = 0.002)]. Additionally, CS/Gl [associated (p = 0.0089) or not with PBM (p = 0.0059)] showed significative lower values for OARSI grade evaluation. Furthermore, CS/GS + PBM decreased IL-1β protein expression (p = 0.0359) and increased IL-10 (p = 0.028) and Col II imunoexpression (p = 0.0204) compared to CG. This study showed that CS/Gl associated with PBM was effective in modulating inflammatory process and preventing the articular tissue degradation in the knees OA rats.

The effect of glucosamine and/or chondroitin sulfate on the progression of knee osteoarthritis: a report from the glucosamine/chondroitin arthritis intervention trial.

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Sawitzke, Allen D; Shi, Helen; Finco, Martha F; Dunlop, Dorothy D; Bingham, Clifton O; Harris, Crystal L; Singer, Nora G; Bradley, John D; Silver, David; Jackson, Christopher G; Lane, Nancy E; Oddis, Chester V; Wolfe, Fred; Lisse, Jeffrey; Furst, Daniel E; Reda, Domenic J; Moskowitz, Roland W; Williams, H James; Clegg, Daniel O

2008-10-01

Osteoarthritis (OA) of the knee causes significant morbidity and current medical treatment is limited to symptom relief, while therapies able to slow structural damage remain elusive. This study was undertaken to evaluate the effect of glucosamine and chondroitin sulfate (CS), alone or in combination, as well as celecoxib and placebo on progressive loss of joint space width (JSW) in patients with knee OA. A 24-month, double-blind, placebo-controlled study, conducted at 9 sites in the United States as part of the Glucosamine/Chondroitin Arthritis Intervention Trial (GAIT), enrolled 572 patients with knee OA who satisfied radiographic criteria (Kellgren/Lawrence [K/L] grade 2 or grade 3 changes and JSW of at least 2 mm at baseline). Patients with primarily lateral compartment narrowing at any time point were excluded. Patients who had been randomized to 1 of the 5 groups in the GAIT continued to receive glucosamine 500 mg 3 times daily, CS 400 mg 3 times daily, the combination of glucosamine and CS, celecoxib 200 mg daily, or placebo over 24 months. The minimum medial tibiofemoral JSW was measured at baseline, 12 months, and 24 months. The primary outcome measure was the mean change in JSW from baseline. The mean JSW loss at 2 years in knees with OA in the placebo group, adjusted for design and clinical factors, was 0.166 mm. No statistically significant difference in mean JSW loss was observed in any treatment group compared with the placebo group. Treatment effects on K/L grade 2 knees, but not on K/L grade 3 knees, showed a trend toward improvement relative to the placebo group. The power of the study was diminished by the limited sample size, variance of JSW measurement, and a smaller than expected loss in JSW. At 2 years, no treatment achieved a predefined threshold of clinically important difference in JSW loss as compared with placebo. However, knees with K/L grade 2 radiographic OA appeared to have the greatest potential for modification by these treatments.

Molecular and biochemical biomarkers for diagnosis and therapy monitorization of Niemann-Pick type C patients.

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Hammerschmidt, Tatiane Grazieli; de Oliveira Schmitt Ribas, Graziela; Saraiva-Pereira, Maria Luiza; Bonatto, Márcia Polese; Kessler, Rejane Gus; Souza, Fernanda Timm Seabra; Trapp, Franciele; Michelin-Tirelli, Kristiane; Burin, Maira Graeff; Giugliani, Roberto; Vargas, Carmen Regla

2018-05-01

Niemann-Pick type C (NP-C), one of 50 inherited lysosomal storage disorders, is caused by NPC protein impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. The clinical manifestations of NP-C include hepatosplenomegaly, neurological and psychiatric symptoms. Current diagnosis for NP-C is based on observation of the accumulated cholesterol in fibroblasts of affected individuals, using an invasive and time expensive test, called Filipin staining. Lately, two metabolites that are markedly increased in NP-C patients are arising as biomarkers for this disease screening: 7-ketocholesterol and cholestane-3β,5α,6β-triol, both oxidized cholesterol products. In this work, we aimed to evaluate the performance of cholestane-3β,5α,6β-triol analysis for the screening and monitoring of NPC patients, correlating it with chitotriosidase levels, Filipin staining and molecular analysis. It was investigated 76 non-treated individuals with NP-C suspicion and also 7 patients with previous NP-C diagnosis under treatment with miglustat, in order to verify the cholestane-3β,5α,6β-triol value as a tool for therapy monitoring. Considering molecular assay as golden standard, it was verified that cholestane-3β,5α,6β-triol analysis presented 88% of sensitivity, 96.08% of specificity, a positive and negative predictive value calculated in 91.67% and 94.23%, respectively, for the diagnosis of NP-C. Chitotriosidase levels were increased in patients with positive molecular analysis for NP-C. For Filipin staining, it was found 1 false positive, 7 false negative and 24 inconclusive cases, showing that this assay has important limitations for NP-C diagnosis. Besides, we found a significant decrease in cholestane-3β,5α,6β-triol concentrations in NP-C patients under therapy with miglustat when compared to non-treated patients. Taken together, the present data show that cholestane-3β,5α,6β-triol analysis has a high potential to be an

Enhancing the intestinal absorption of low molecular weight chondroitin sulfate by conjugation with α-linolenic acid and the transport mechanism of the conjugates.

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Xiao, Yuliang; Li, Pingli; Cheng, Yanna; Zhang, Xinke; Sheng, Juzheng; Wang, Decai; Li, Juan; Zhang, Qian; Zhong, Chuanqing; Cao, Rui; Wang, Fengshan

2014-04-25

The purpose of this report was to demonstrate the effect of amphiphilic polysaccharides-based self-assembling micelles on enhancing the oral absorption of low molecular weight chondroitin sulfate (LMCS) in vitro and in vivo, and identify the transepithelial transport mechanism of LMCS micelles across the intestinal barrier. α-Linolenic acid-low molecular weight chondroitin sulfate polymers(α-LNA-LMCS) were successfully synthesized, and characterized by FTIR, (1)HNMR, TGA/DSC, TEM, laser light scattering and zeta potential. The significant oral absorption enhancement and elimination half-life (t₁/₂) extension of LNA-LMCS2 in rats were evidenced by intragastric administration in comparison with CS and LMCS. Caco-2 transport studies demonstrated that the apparent permeability coefficient (Papp) of LNA-LMCS2 was significantly higher than that of CS and LMCS (p<0.001), and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, α-LNA-LMCS micelles accumulated around the cell membrane and intercellular space observed by confocal laser scanning microscope (CLSM). Furthermore, evident alterations in the F-actin cytoskeleton were detected by CLSM observation following the treatment of the cell monolayers with α-LNA-LMCS micelles, which further certified the capacity of α-LNA-LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Therefore, LNA-LMCS2 with low cytotoxicity and high bioavailability might be a promising substitute for CS in clinical use, such as treating osteoarthritis, atherosclerosis, etc. Copyright © 2014 Elsevier B.V. All rights reserved.

Inhibition of synthesis of heparan sulfate by selenate: Possible dependence on sulfation for chain polymerization

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Dietrich, C.P.; Nader, H.B.; Buonassisi, V.; Colburn, P.

1988-01-01

Selenate, a sulfation inhibitor, blocks the synthesis of heparan sulfate and chondroitin sulfate by cultured endothelial cells. In contrast, selenate does not affect the production of hyaluronic acid, a nonsulfated glycosaminoglycan. No differences in molecular weight, [ 3 H]glucosamine/[ 35 S]sulfuric acid ratios, or disaccharide composition were observed when the heparan sulfate synthesized by selenate-treated cells was compared with that of control cells. The absence of undersulfated chains in preparations from cultures exposed to selenate supports the concept that, in the intact cell, the polymerization of heparan sulfate might be dependent on the sulfation of the saccharide units added to the growing glycosaminoglycan chain

Polyethylene glycol-conjugated chondroitin sulfate A derivative nanoparticles for tumor-targeted delivery of anticancer drugs.

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Lee, Jae-Young; Park, Ju-Hwan; Lee, Jeong-Jun; Lee, Song Yi; Chung, Suk-Jae; Cho, Hyun-Jong; Kim, Dae-Duk

2016-10-20

Polyethylene glycol (PEG)-decorated chondroitin sulfate A-deoxycholic acid (CSD) nanoparticles (NPs) were fabricated for the selective delivery of doxorubicin (DOX) to ovarian cancer. CSD-PEG was synthesized via amide bond formation between the NH2 group of methoxypolyethylene glycol amine and the COOH group of CSD. CSD-PEG/DOX NPs with a 247nm mean diameter, negative zeta potential, and >90% drug encapsulation efficiency were prepared. Sustained and pH-dependent DOX release profiles from CSD-PEG NPs were observed in dissolution tests. Endocytosis of NPs by SKOV-3 cells (CD44 receptor-positive human ovarian cancer cells), based on the CSA-CD44 receptor interaction, was determined by flow cytometry and confocal laser scanning microscopy (CLSM) studies. PEGylation of NPs also resulted in reduced drug clearance (CL) in vivo and improved relative bioavailability, compared to non-PEGylated NPs, as determined by the pharmacokinetic study performed after intravenous administration in rats. Developed CSD-PEG NPs can be a promising delivery vehicle for the therapy of CD44 receptor-expressing ovarian cancers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Alteration of gene expression profile in Niemann-Pick type C mice correlates with tissue damage and oxidative stress.

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Mary C Vázquez

Full Text Available BACKGROUND: Niemann-Pick type C disease (NPC is a neurovisceral lipid storage disorder mainly characterized by unesterified cholesterol accumulation in lysosomal/late endosomal compartments, although there is also an important storage for several other kind of lipids. The main tissues affected by the disease are the liver and the cerebellum. Oxidative stress has been described in various NPC cells and tissues, such as liver and cerebellum. Although considerable alterations occur in the liver, the pathological mechanisms involved in hepatocyte damage and death have not been clearly defined. Here, we assessed hepatic tissue integrity, biochemical and oxidative stress parameters of wild-type control (Npc1(+/+; WT and homozygous-mutant (Npc1(-/-; NPC mice. In addition, the mRNA abundance of genes encoding proteins associated with oxidative stress, copper metabolism, fibrosis, inflammation and cholesterol metabolism were analyzed in livers and cerebella of WT and NPC mice. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed various oxidative stress parameters in the liver and hepatic and cerebellum gene expression in 7-week-old NPC1-deficient mice compared with control animals. We found signs of inflammation and fibrosis in NPC livers upon histological examination. These signs were correlated with increased levels of carbonylated proteins, diminished total glutathione content and significantly increased total copper levels in liver tissue. Finally, we analyzed liver and cerebellum gene expression patterns by qPCR and microarray assays. We found a correlation between fibrotic tissue and differential expression of hepatic as well as cerebellar genes associated with oxidative stress, fibrosis and inflammation in NPC mice. CONCLUSIONS/SIGNIFICANCE: In NPC mice, liver disease is characterized by an increase in fibrosis and in markers associated with oxidative stress. NPC is also correlated with altered gene expression, mainly of genes involved in oxidative stress

Chronic cyclodextrin treatment of murine Niemann-Pick C disease ameliorates neuronal cholesterol and glycosphingolipid storage and disease progression.

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Cristin D Davidson

2009-09-01

Full Text Available Niemann-Pick type C (NPC disease is a fatal neurodegenerative disorder caused most commonly by a defect in the NPC1 protein and characterized by widespread intracellular accumulation of unesterified cholesterol and glycosphingolipids (GSLs. While current treatment therapies are limited, a few drugs tested in Npc1(-/- mice have shown partial benefit. During a combination treatment trial using two such compounds, N-butyldeoxynojirimycin (NB-DNJ and allopregnanolone, we noted increased lifespan for Npc1(-/- mice receiving only 2-hydroxypropyl-beta-cyclodextrin (CD, the vehicle for allopregnanolone. This finding suggested that administration of CD alone, but with greater frequency, might provide additional benefit.Administration of CD to Npc1(-/- mice beginning at either P7 or P21 and continuing every other day delayed clinical onset, reduced intraneuronal cholesterol and GSL storage as well as free sphingosine accumulation, reduced markers of neurodegeneration, and led to longer survival than any previous treatment regime. We reasoned that other lysosomal diseases characterized by cholesterol and GSL accumulation, including NPC disease due to NPC2 deficiency, GM1 gangliosidosis and mucopolysaccharidosis (MPS type IIIA, might likewise benefit from CD treatment. Treated Npc2(-/- mice showed benefits similar to NPC1 disease, however, mice with GM1 gangliosidosis or MPS IIIA failed to show reduction in storage.Treatment with CD delayed clinical disease onset, reduced intraneuronal storage and secondary markers of neurodegeneration, and significantly increased lifespan of both Npc1(-/- and Npc2(-/- mice. In contrast, CD failed to ameliorate cholesterol or glycosphingolipid storage in GM1 gangliosidosis and MPS IIIA disease. Understanding the mechanism(s by which CD leads to reduced neuronal storage may provide important new opportunities for treatment of NPC and related neurodegenerative diseases characterized by cholesterol dyshomeostasis.

Recent Advances in the Diagnosis and Treatment of Niemann-Pick Disease Type C in Children: A Guide to Early Diagnosis for the General Pediatrician

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Hanna Alobaidy

2015-01-01

Full Text Available Niemann-Pick disease (NP-C is a lysosomal storage disease in which impaired intracellular lipid transport leads to accumulation of cholesterol and glycosphingolipids in various neurovisceral tissues. It is an autosomal recessive disorder, caused by mutations in the NPC1 or NPC2 genes. The clinical spectrum is grouped by the age of onset and onset of neurological manifestation: pre/perinatal; early infantile; late infantile; and juvenile periods. The NP-C Suspicion Index (SI screening tool was developed to identify suspected patients with this disease. It is especially good at recognizing the disease in patients older than four years of age. Biochemical tests involving genetic markers and Filipin staining of skin fibroblast are being employed to assist diagnosis. Therapy is mostly supportive and since 2009, the first specific therapy approved for use was Miglustat (Zavesca aimed at stabilizing the rate of progression of neurological manifestation. The prognosis correlates with age at onset of neurological signs; patients with early onset form progress faster. The NP-C disease has heterogeneous neurovisceral manifestations. A SI is a screening tool that helps in diagnostic process. Filipin staining test is a specific biomarker diagnostic test. Miglustat is the first disease-specific therapy.

Iduronic acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells.

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Barbara Bartolini

Full Text Available Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA, catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1-/- aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK and phospho-FAK (pFAK was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.

Effect of introduction of chondroitin sulfate into polymer-peptide conjugate responding to intracellular signals

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Tomiyama, Tetsuro; Toita, Riki; Kang, Jeong-Hun; Koga, Haruka; Shiosaki, Shujiro; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki

2011-09-01

We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.

Effect of the chelation of metal cation on the antioxidant activity of chondroitin sulfates.

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Ajisaka, Katsumi; Oyanagi, Yutaka; Miyazaki, Tatsuo; Suzuki, Yasuhiro

2016-06-01

The antioxidant potencies of chondroitin sulfates (CSs) from shark cartilage, salmon cartilage, bovine trachea, and porcine intestinal mucosa were compared by three representative methods for the measurement of the antioxidant activity; DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity. CSs from salmon cartilage and bovine trachea showed higher potency in comparison with CSs from shark cartilage and porcine intestinal mucosa. Next, CS from salmon cartilage chelating with Ca(2+), Mg(2+), Mn(2+), or Zn(2+) were prepared, and their antioxidant potencies were compared. CS chelating with Ca(2+) or Mg(2+) ions showed rather decreased DPPH radical scavenging activity in comparison with CS of H(+) form. In contrast, CS chelating with Ca(2+) or Mg(2+) ion showed remarkably enhanced superoxide radical scavenging activity than CS of H(+) or Na(+) form. Moreover, CS chelating with divalent metal ions, Ca(2+), Mg(2+), Mn(2+), or Zn(2+), showed noticeably higher hydroxyl radical scavenging activity than CS of H(+) or Na(+) form. The present results revealed that the scavenging activities of, at least, superoxide radical and hydroxyl radical were enhanced by the chelation with divalent metal ions.

Novel bioadhesive polymers as intra-articular agents: Chondroitin sulfate-cysteine conjugates.

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Suchaoin, Wongsakorn; Bonengel, Sonja; Griessinger, Julia Anita; Pereira de Sousa, Irene; Hussain, Shah; Huck, Christian W; Bernkop-Schnürch, Andreas

2016-04-01

The aim of this study was to generate and characterize a chondroitin sulfate-cysteine conjugate (CS-cys) as a novel bioadhesive agent for intra-articular use. Mucoadhesive properties of synthesized CS-cys were investigated by rheological measurement of polymer-mucus mixture and rotating cylinder method, while bioadhesive features of CS-cys on porcine articular cartilage were evaluated via tensile studies. Thiolation was achieved by attachment of l-cysteine to CS via amide bond formation mediated by carbodiimide as a coupling reagent. The conjugate exhibited 421.17±35.14 μmol free thiol groups per gram polymer. The reduced CS-cys displayed 675.09±39.67 μmol free thiol groups per gram polymer after disulfide bonds reduction using tris(2-carboxyethyl)phosphine hydrochloride. The increase in dynamic viscosity of thiolated CS due to oxidative disulfide bond formation was demonstrated using capillary viscometer. The combination of CS-cys and mucus led to 4.57-fold increase in dynamic viscosity in comparison with mucus control. Furthermore, adhesion time to porcine mucosa of CS-cys-based test disk was enhanced by 2.48-fold compared to unmodified CS as measured by rotating cylinder method suggesting the interaction between thiomers and mucus gel layer via disulfide bonds formation. Tensile studies of thiolated CS on porcine articular cartilage showed 5.37- and 1.76-fold increase in the total work of adhesion and the maximum detachment force, respectively, in comparison with unmodified CS indicating bioadhesive features of CS-cys. Cytotoxicity of CS-cys was assessed in Caco-2 cells and rat primary articular chondrocytes using MTT and LDH release assay, thereby showing the safety of CS-cys at a concentration of 0.25% (w/v) in Caco-2 cells. Furthermore, 0.1% of CS-cys was found non-toxic to rat primary articular chondrocytes. According to these results, CS-cys provides improved bioadhesive properties that might be useful as an intra-articular agent for treatment of

Mitochondrial G8292A and C8794T mutations in patients with Niemann-Pick disease type C.

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Masserrat, Abbas; Sharifpanah, Fatemeh; Akbari, Leila; Tonekaboni, Seyed Hasan; Karimzadeh, Parvaneh; Asharafi, Mahmood Reza; Mazouei, Safoura; Sauer, Heinrich; Houshmand, Massoud

2018-07-01

Niemann-Pick disease type C (NP-C) is a neurovisceral lipid storage disorder. At the cellular level, the disorder is characterized by accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system. NP-C is transmitted in an autosomal recessive manner and is caused by mutations in either the NPC1 (95% of families) or NPC2 gene. The estimated disease incidence is 1 in 120,000 live births, but this likely represents an underestimate, as the disease may be under-diagnosed due to its highly heterogeneous presentation. Variants of adenosine triphosphatase (ATPase) subunit 6 and ATPase subunit 8 ( ATPase6/8 ) in mitochondrial DNA (mtDNA) have been reported in different types of genetic diseases including NP-C. In the present study, the blood samples of 22 Iranian patients with NP-C and 150 healthy subjects as a control group were analyzed. The DNA of the blood samples was extracted by the salting out method and analyzed for ATPase6/8 mutations using polymerase chain reaction sequencing. Sequence variations in mitochondrial genome samples were determined via the Mitomap database. Analysis of sequencing data confirmed the existence of 11 different single nucleotide polymorphisms (SNPs) in patients with NP-C1. One of the most prevalent polymorphisms was the A8860G variant, which was observed in both affected and non-affected groups and determined to have no significant association with NP-C incidence. Amongst the 11 polymorphisms, only one was identified in the ATPase8 gene, while 9 including A8860G were observed in the ATPase6 gene. Furthermore, two SNPs, G8292A and C8792A, located in the non-coding region of mtDNA and the ATPase6 gene, respectively, exhibited significantly higher prevalence rates in NP-C1 patients compared with the control group (PC disease. In addition, the mitochondrial SNPs identified maybe pathogenic mutations involved in the development and prevalence of NP-C. Furthermore, these results suggest a higher occurrence of

Loss of Niemann-Pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.

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Sayali S Dixit

Full Text Available Niemann-Pick Type C (NPC disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.

Reconstrução do ligamento cruzado cranial em cães, associado ou não ao sulfato de condroitina Cranial cruciate ligament reconstruction in dogs associated or not to chondroitin sulfate

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F. Biasi

2005-08-01

Full Text Available Avaliou-se o efeito da reconstrução do ligamento cruzado cranial, associado ou não ao sulfato de condroitina, na evolução da osteoartrite induzida experimentalmente em cães. Vinte cães hígidos, sem raça definida, machos e fêmeas, com peso corpóreo entre 19 e 25kg, foram submetidos à desmotomia do ligamento cruzado cranial. Trinta dias após, foram separados em dois grupos de 10 animais. Um grupo foi submetido à reconstrução do ligamento cruzado com uso de aloenxerto de ligamento patelar congelado, o outro não. Trinta e um dias após a desmotomia, cada grupo foi dividido em dois subgrupos de cinco animais. Um recebeu sulfato de condroitina, o outro não. Os cães foram avaliados clínica e radiograficamente antes da desmotomia e aos 30, 60 e 90 dias após a desmotomia. No último momento foram realizados exames macro e microscópico. Nos cães submetidos somente à desmotomia e tratados com sulfato de condroitina houve redução na progressão das alterações ósseas, ao exame radiográfico. A reconstrução do ligamento cruzado cranial melhorou a função do membro e, quando associada ao sulfato de condroitina, houve melhor resposta. Não houve diferença entre os subgrupos quanto aos exames macro e microscópico.The effect of cranial cruciate ligament reconstruction, associated or not to chondroitin sulfate, on the evolution of experimentally induced osteoarthritis in dogs was studied. Twenty healthy mixed dogs, weighing between 19 and 25kg were submitted to cranial cruciate desmotomy. Thirty days later, the animals were divided into two groups with ten dogs each. One was submitted to cranial cruciate ligament reconstruction using frozen patellar tendon allograft and the other received no surgical treatment. Thirty one days after desmotomy, each group was divided into two subgroups with five animals each. One subgroup for each group received chondroitin sulfate and the other received no medical treatment. The dogs were

Extraction of Glycosaminoglycans Containing Glucosamine and Chondroitin Sulfate from Chicken Claw Cartilage

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Tri Dewanti Widyaningsih

2017-12-01

Full Text Available Chicken cartilage (claw is a waste of chicken cuts which are widely available in Indonesia. Cartilage part of chicken claw becomes a potential source of chondroitin sulfate (CS and glucosamine (GS. This study aims to determine the most optimal extraction methods of CS and GS from cartilage of chicken claw. Various types of extraction methods used in this study are taken from the extraction by using boiling water (2 and 2.5 hours, acetic acid (7 and 17 hours, as well as proteolysis by papain (24 and 48 hours. Parameters observed include chemical characteristics of powdered cartilage of chicken claw as well as CS and GS levels in powdered cartilage of chicken claw extract. The results of this research show that the levels of CS and GS of chicken claw cartilage powder were 2.17% and 13%. Meanwhile, the highest GS level was obtained from the extraction with water treatment for 2.5 hours which was 8.1%. The treatment and duration of extraction will significantly affect the number of GS which was produced. The highest content of CS was obtained from the extraction with the enzyme treatment for 48 hours which was 2.47%. The best treatment is the extraction with water treatment for 2.5 hours which were the extracts with GS levels of 8.1% and 2.03% CS was selected through the analysis of multiple attribute.

Reduction-sensitive micelles self-assembled from amphiphilic chondroitin sulfate A-deoxycholic acid conjugate for triggered release of doxorubicin.

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Liu, Hongxia; Wu, Shuqin; Yu, Jingmou; Fan, Dun; Ren, Jin; Zhang, Lei; Zhao, Jianguo

2017-06-01

Reduction-sensitive chondroitin sulfate A (CSA)-based micelles were developed. CSA was conjugated with deoxycholic acid (DOCA) via a disulfide linkage. The bioreducible conjugate (CSA-ss-DOCA) can form self-assembled micelles in aqueous medium. The critical micelle concentration (CMC) of CSA-ss-DOCA conjugate is 0.047mg/mL, and its mean diameter is 387nm. The anticancer drug doxorubicin (DOX) was chosen as a model drug, and was effectively encapsulated into the micelles with high loading efficiency. Reduction-sensitive micelles and reduction-insensitive control micelles displayed similar DOX release behavior in phosphate buffered saline (PBS, pH7.4). Notably, DOX release from the reduction-sensitive micelles in vitro was accelerated in the presence of 20mM glutathione-containing PBS environment. Moreover, DOX-loaded CSA-ss-DOCA (CSA-ss-DOCA/DOX) micelles exhibited intracellular reduction-responsive characteristics in human gastric cancer HGC-27 cells determined by confocal laser scanning microscopy (CLSM). Furthermore, CSA-ss-DOCA/DOX micelles demonstrated higher antitumor efficacy than reduction-insensitive control micelles in HGC-27 cells. These results suggested that reduction-sensitive CSA-ss-DOCA micelles had the potential as intracellular targeted carriers of anticancer drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficacy of a fixed combination of 0.09 % xanthan gum/0.1 % chondroitin sulfate preservative free vs polyethylene glycol/propylene glycol in subjects with dry eye disease: a multicenter randomized controlled trial.

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Pérez-Balbuena, Ana L; Ochoa-Tabares, Juan C; Belalcazar-Rey, Sandra; Urzúa-Salinas, Cristian; Saucedo-Rodríguez, Laura R; Velasco-Ramos, Regina; Suárez-Sánchez, Raúl G; Rodríguez-Carrizalez, Adolfo D; Oregón-Miranda, Aldo A

2016-09-20

Dry eye disease (DED) is multifactorial, affecting 5-34 % of the global adult population and reducing quality of life. The artificial tears or lubricants are the therapy most used for the treatment of DED, due to their low side effect profile, which attempt to modify the properties of the tear film. The aim of the present study was to evaluate the clinical efficacy of a fixed combination of xanthan gum and chondroitin sulfate preservative free on the ocular surface of patients with dry eye disease during 60 days of intervention. A phase III, double-blind, masked, controlled, multicenter, clinical trial of 148 subjects, randomized to either a fixed combination of xanthan gum 0.09 % and chondroitin sulfate 0.1 % (XG/CS) ophthalmic solution (n = 76) or a fixed combination of polyethylene glycol 400 0.4 % and propylene glycol 0.3 % (PEG/PG) (n = 72). Subjects self-dosed four times daily during 60 days. Follow-up was set on days 2, 7, 15, 30 and 60. Assessments of anterior/posterior segment ocular signs were performed. The outcome measures included Schirmer test, tear film break-up time and OSDI score. Security variables included intraocular pressure, lisamine green and fluorescein ocular surface stains. The primary efficacy endpoints were similar between groups at baseline. After intervention time Schirmer test increased in both groups compared to baseline, XG/CS (6.4 ± 2.2 vs 11.0 ± 6.6; p = 0.002) and PEG/PG (6.5 ± 2.5 vs 10.5 ± 5.6; p = 0.019) respectively. Similar results were reported in the tear film break-up time in XG/CS (5.5 ± 2.1 vs 7.4 ± 2.9; p = 0.027) and PEG/PG (5.2 ± 2.0 vs 7.4 ± 2.7; p = 0.046) respectively. The OSDI score decreased to normal values in both groups, XG/CS (19.3 ± 7.4 vs 7.3 ± 5.9; p = 0.001) and PEG/PG (19.3 ± 7.5 vs 7.9 ± 8.2; p = 0.001) respectively. There was no significant difference between treatments for any parameter. Moreover, both

Miglustat therapy in the French cohort of paediatric patients with Niemann-Pick disease type C

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Héron Bénédicte

2012-06-01

Full Text Available Abstract Background Niemann-Pick disease type C (NP-C is a rare neurovisceral lysosomal lipid storage disease characterized by progressive neurological deterioration. Published data on the use of miglustat in paediatric patients in clinical practice settings are limited. We report findings from a prospective open-label study in the French paediatric NP-C cohort. Methods Data on all paediatric NP-C patients treated with miglustat in France between October 2006 and December 2010 were compiled. All patients had a confirmed diagnosis of NP-C, and received miglustat therapy according to manufacturer’s recommendations. Pre-treatment and follow-up assessments were conducted according to a standardized protocol. Results Twenty children were enrolled; 19 had NPC1 gene mutations and 1 had NPC2 gene mutations. The median age at diagnosis was 1.5 years, and the median age at miglustat initiation was 6.0 years. Eight NPC1 patients had the early-infantile, eight had the late-infantile, and three had the juvenile-onset forms of NP-C. A history of hepatosplenomegaly and/or other cholestatic symptoms was recorded in all 8 early-infantile onset patients, 3/8 late-infantile patients, and 1/3 juvenile onset patients. Brain imaging indicated white matter abnormalities in most patients. The median (range duration of miglustat therapy was 1.3 (0.6–2.3 years in early-infantile, 1.0 (0.8–5.0 year in late-infantile, and 1.0 (0.6–2.5 year in juvenile onset patients. NP-C disability scale scores indicated either stabilization or improvement of neurological manifestations in 1/8, 6/8, and 1/3 NPC1 patients in these subgroups, respectively. There were no correlations between brain imaging findings and disease course. Mild-to-moderate gastrointestinal disturbances were frequent during the first 3 months of miglustat therapy, but were easily managed with dietary modifications and/or anti-propulsive medication. Conclusions Miglustat can improve or stabilize neurological

Mitotic spindle defects and chromosome mis-segregation induced by LDL/cholesterol-implications for Niemann-Pick C1, Alzheimer's disease, and atherosclerosis.

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Antoneta Granic

Full Text Available Elevated low-density lipoprotein (LDL-cholesterol is a risk factor for both Alzheimer's disease (AD and Atherosclerosis (CVD, suggesting a common lipid-sensitive step in their pathogenesis. Previous results show that AD and CVD also share a cell cycle defect: chromosome instability and up to 30% aneuploidy-in neurons and other cells in AD and in smooth muscle cells in atherosclerotic plaques in CVD. Indeed, specific degeneration of aneuploid neurons accounts for 90% of neuronal loss in AD brain, indicating that aneuploidy underlies AD neurodegeneration. Cell/mouse models of AD develop similar aneuploidy through amyloid-beta (Aß inhibition of specific microtubule motors and consequent disruption of mitotic spindles. Here we tested the hypothesis that, like upregulated Aß, elevated LDL/cholesterol and altered intracellular cholesterol homeostasis also causes chromosomal instability. Specifically we found that: 1 high dietary cholesterol induces aneuploidy in mice, satisfying the hypothesis' first prediction, 2 Niemann-Pick C1 patients accumulate aneuploid fibroblasts, neurons, and glia, demonstrating a similar aneugenic effect of intracellular cholesterol accumulation in humans 3 oxidized LDL, LDL, and cholesterol, but not high-density lipoprotein (HDL, induce chromosome mis-segregation and aneuploidy in cultured cells, including neuronal precursors, indicating that LDL/cholesterol directly affects the cell cycle, 4 LDL-induced aneuploidy requires the LDL receptor, but not Aß, showing that LDL works differently than Aß, with the same end result, 5 cholesterol treatment disrupts the structure of the mitotic spindle, providing a cell biological mechanism for its aneugenic activity, and 6 ethanol or calcium chelation attenuates lipoprotein-induced chromosome mis-segregation, providing molecular insights into cholesterol's aneugenic mechanism, specifically through its rigidifying effect on the cell membrane, and potentially explaining why ethanol

Chondroitin sulfate iron colloid-enhanced MR imaging of hepatocellular carcinoma; Correlation between histologic grade and detectability

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Kamba, Masayuki; Suto, Yuji; Kodama, Fumiko; Kato, Terumi; Ohta, Yoshio; Horie, Yasushi; Hamazoe, Ryuichi; Kawasaki, Hironaka (Tottori Univ., Yonago (Japan). School of Medicine)

1994-03-01

We applied chondroitin sulfate iron colloid (CSIC) as an MR contrast agent to detect hepatocellular carcinoma (HCC). The MR and pathologic findings of 25 HCCs in 21 patients were analyzed. MR imaging was performed with a superconducting system operating at 1.5 T. Proton density-weighted (PDW), T[sub 2]-weighted (T[sub 2]W) and T[sub 1]-weighted (T[sub 1]W) images were obtained before and after an intravenous injection of 23.6 [mu]mol Fe/kg of CSIC. In moderately to poorly differentiated and moderately differentiated HCCs (n=15), all the lesions except a 5-mm satellite nodule were detectable with unenhanced T[sub 2]W images as well as CSIC-enhanced PDW, T[sub 2]W and T[sub 1]W images. In well to moderately differentiated HCCs (n=6), two to four lesions were detectable with unenhanced images. All the lesions except a 3-mm satellite nodule were detectable with CSIC-enhanced PDW, T[sub 2]W and T[sub 1]W images. In well differentiated HCCs (n=4), one or two lesions were detectable with unenhanced images. All the lesions were detectable with CSIC-enhanced T[sub 1]W images, while only two lesions were detectable with CSIC-enhanced PDW or T[sub 2]W images. CSIC administration improves detection rates, and is especially useful in detecting small foci of well to moderately or well differentiated HCC. (author).

Hydrogels based on chemically modified poly(vinyl alcohol (PVA-GMA and PVA-GMA/chondroitin sulfate: Preparation and characterization

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E. C. Muniz

2012-05-01

Full Text Available This work reports the preparation of hydrogels based on PVA-GMA, PVA-GMA is poly(vinyl alcohol (PVA functionalized with vinyl groups from glycidyl methacrylate (GMA, and on PVA-GMA with different content of chondroitin sulfate (CS. The degrees of swelling of PVA-GMA and PVA-GMA/CS hydrogels were evaluated in distilled water and the swelling kinetics was performed in simulated gastric and intestinal fluids (SGF and SIF. PVA-GMA and PVAGMA/CS hydrogels demonstrated to be resistant on SGF and SIF fluids. The elastic modulus, E, of swollen-hydrogels were determined through compressive tests and, according to the obtained results, the hydrogels presented good mechanical properties. At last, the presence of CS enhances the hydrogel cell compatibility as gathered by cytotoxicity assays. It was concluded that the hydrogels prepared through this work presented characteristics that allow them to be used as biomaterial, as a carrier in drug delivery system or to act as scaffolds in tissue engineering as well.

Chondroitin sulfate iron colloid-enhanced MR imaging in patients with hepatocellular carcinoma. Comparison with CT during arterial portography

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Kamba, Masayuki [Dept. of Radiology, Tottori Univ., Yonago (Japan); Suto, Y. [Dept. of Radiology, Tottori Univ., Yonago (Japan); Kato, T. [Dept. of Radiology, Tottori Univ., Yonago (Japan)

1994-11-01

Chondroitin sulfate iron colloid (CSIC) was used as an MR contrast agent for the detection of hepatocellular carcinoma (HCC). The findings of 25 surgically confirmed HCCs in 19 patients were retrospectively analyzed. T1-, T2- and proton density-weighted spin echo MR images were obtained before and after i.v. injection of 23.6 {mu}M Fe/kg of CSIC. Unenhanced and CSIC-enhanced MR images and images obtained by CT during arterial protography (CT-AP) were correlated with surgical pathology findings. The sensitivities of CSIC-enhanced and unenhanced MR imaging, and CT-AP were 92%, 80%, and 88%, respectively. No significant differences were noted. Portal flow abnormalities demonstrated by CT-AP did not affect the detection of HCC by CSIC-enhanced MR imaging. CSIC-enhancement at MR imaging was a disadvantage in the detection of lesions less than 1 cm in diameter. CSIC-enhanced MR imaging is a supplemental method for the detection of HCC. (orig.).

Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

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Rottiers, P; Verfaillie, T; Contreras, R; Revets, H; Desmedt, M; Dooms, H; Fiers, W; Grooten, J

1998-11-09

Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.

Alterations in gene expression in mutant amyloid precursor protein transgenic mice lacking Niemann-Pick type C1 protein.

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Mahua Maulik

Full Text Available Niemann-Pick type C (NPC disease, a rare autosomal recessive disorder caused mostly by mutation in NPC1 gene, is pathologically characterized by the accumulation of free cholesterol in brain and other tissues. This is accompanied by gliosis and loss of neurons in selected brain regions, including the cerebellum. Recent studies have shown that NPC disease exhibits intriguing parallels with Alzheimer's disease, including the presence of neurofibrillary tangles and increased levels of amyloid precursor protein (APP-derived β-amyloid (Aβ peptides in vulnerable brain neurons. To evaluate the role of Aβ in NPC disease, we determined the gene expression profile in selected brain regions of our recently developed bigenic ANPC mice, generated by crossing APP transgenic (Tg mice with heterozygous Npc1-deficient mice. The ANPC mice exhibited exacerbated neuronal and glial pathology compared to other genotypes [i.e., APP-Tg, double heterozygous (Dhet, Npc1-null and wild-type mice]. Analysis of expression profiles of 86 selected genes using real-time RT-PCR arrays showed a wide-spectrum of alterations in the four genotypes compared to wild-type controls. The changes observed in APP-Tg and Dhet mice are limited to only few genes involved mostly in the regulation of cholesterol metabolism, whereas Npc1-null and ANPC mice showed alterations in the expression profiles of a number of genes regulating cholesterol homeostasis, APP metabolism, vesicular trafficking and cell death mechanism in both hippocampus and cerebellum compared to wild-type mice. Intriguingly, ANPC and Npc1-null mice, with some exceptions, exhibited similar changes, although more genes were differentially expressed in the affected cerebellum than the relatively spared hippocampus. The altered gene profiles were found to match with the corresponding protein levels. These results suggest that lack of Npc1 protein can alter the expression profile of selected transcripts as well as proteins, and

Green synthesis of chondroitin sulfate-capped silver nanoparticles: characterization and surface modification.

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Cheng, Kuang-ming; Hung, Yao-wen; Chen, Cheng-cheung; Liu, Cheng-che; Young, Jenn-jong

2014-09-22

A one-step route for the green synthesis of highly stable and nanosized silver metal particles with narrow distribution is reported. In this environmentally friendly synthetic method, silver nitrate was used as silver precursor and biocompatible chondroitin sulfate (ChS) was used as both reducing agent and stabilizing agent. The reaction was carried out in a stirring aqueous medium at the room temperature without any assisted by microwave, autoclave, laser irradiation, γ-ray irradiation or UV irradiation. The transparent colorless solution was converted to the characteristics light red then deep red-brown color as the reaction proceeds, indicating the formation of silver nanoparticles (Ag NPs). The Ag NPs were characterized by UV-visible spectroscopy (UV-vis), photon correlation spectroscopy, laser Doppler anemometry, transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FT-IR). The results demonstrated that the obtained metallic nanoparticles were Ag NPs capped with ChS. In this report, dynamic light scattering (DLS) was used as a routinely analytical tool for measuring size and distribution in a liquid environment. The effects of the reaction time, reaction temperature, concentration and the weight ratio of ChS/Ag+ on the particle size and zeta potential were investigated. The TEM image clearly shows the morphology of the well-dispersed ChS-capped Ag NPs are spherical in shape, and the average size (propyl] chitosan chloride (HTCC) were prepared by an ionic gelation method and the surface charge of Ag NPs was switched from negative to positive. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quantitative Analysis of the Proteome Response to the Histone Deacetylase Inhibitor (HDACi) Vorinostat in Niemann-Pick Type C1 disease.

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Subramanian, Kanagaraj; Rauniyar, Navin; Lavalleé-Adam, Mathieu; Yates, John R; Balch, William E

2017-11-01

Niemann-Pick type C (NPC) disease is an inherited, progressive neurodegenerative disorder principally caused by mutations in the NPC1 gene. NPC disease is characterized by the accumulation of unesterified cholesterol in the late endosomes (LE) and lysosomes (Ly) (LE/Ly). Vorinostat, a histone deacetylase inhibitor (HDACi), restores cholesterol homeostasis in fibroblasts derived from NPC patients; however, the exact mechanism by which Vorinostat restores cholesterol level is not known yet. In this study, we performed comparative proteomic profiling of the response of NPC1 I1061T fibroblasts to Vorinostat. After stringent statistical criteria to filter identified proteins, we observed 202 proteins that are differentially expressed in Vorinostat-treated fibroblasts. These proteins are members of diverse cellular pathways including the endomembrane dependent protein folding-stability-degradation-trafficking axis, energy metabolism, and lipid metabolism. Our study shows that treatment of NPC1 I1061T fibroblasts with Vorinostat not only enhances pathways promoting the folding, stabilization and trafficking of NPC1 (I1061T) mutant to the LE/Ly, but alters the expression of lysosomal proteins, specifically the lysosomal acid lipase (LIPA) involved in the LIPA->NPC2->NPC1 based flow of cholesterol from the LE/Ly lumen to the LE/Ly membrane. We posit that the Vorinostat may modulate numerous pathways that operate in an integrated fashion through epigenetic and post-translational modifications reflecting acetylation/deacetylation balance to help manage the defective NPC1 fold, the function of the LE/Ly system and/or additional cholesterol metabolism/distribution pathways, that could globally contribute to improved mitigation of NPC1 disease in the clinic based on as yet uncharacterized principles of cellular metabolism dictating cholesterol homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential response of the liver to bile acid treatment in a mouse model of Niemann-Pick disease type C [version 2; referees: 2 approved, 1 not approved

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Elena-Raluca Nicoli

2018-04-01

Full Text Available Niemann-Pick disease type C (NPC disease is a neurodegenerative lysosomal storage disease caused by mutations in the NPC1 or NPC2 genes. Liver disease is also a common feature of NPC that can present as cholestatic jaundice in the neonatal period. Liver enzymes can remain elevated above the normal range in some patients as they age. We recently reported suppression of the P450 detoxification system in a mouse model of NPC disease and also in post-mortem liver from NPC patients. We demonstrated the ability of the hydrophobic bile acid ursodeoxycholic acid (UDCA (3α, 7β-dihydroxy-5β-cholanic acid to correct the P450 system suppression. UDCA is used to treat several cholestatic disorders and was tested in NPC due to the P450 system being regulated by bile acids. Here, we compare the effect of UDCA and cholic acid (CA, another bile acid, in the NPC mouse model. We observed unexpected hepatotoxicity in response to CA treatment of NPC mice. No such hepatotoxicity was associated with UDCA treatment. These results suggest that CA treatment is contraindicated in NPC patients, whilst supporting the use of UDCA as an adjunctive therapy in NPC patients.

212Pb-Labeled Antibody 225.28 Targeted to Chondroitin Sulfate Proteoglycan 4 for Triple-Negative Breast Cancer Therapy in Mouse Models

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Benjamin B. Kasten

2018-03-01

Full Text Available Triple-negative breast cancer (TNBC is an aggressive subtype of breast cancer with a poor prognosis. There is a clinical need for effective, targeted therapy strategies that destroy both differentiated TNBC cells and TNBC cancer initiating cells (CICs, as the latter are implicated in the metastasis and recurrence of TNBC. Chondroitin sulfate proteoglycan 4 (CSPG4 is overexpressed on differentiated tumor cells and CICs obtained from TNBC patient specimens, suggesting that CSPG4 may be a clinically relevant target for the imaging and therapy of TNBC. The purpose of this study was to determine whether α-particle radioimmunotherapy (RIT targeting TNBC cells using the CSPG4-specific monoclonal antibody (mAb 225.28 as a carrier was effective at eliminating TNBC tumors in preclinical models. To this end, mAb 225.28 labeled with 212Pb (212Pb-225.28 as a source of α-particles for RIT was used for in vitro Scatchard assays and clonogenic survival assays with human TNBC cells (SUM159 and 2LMP grown as adherent cells or non-adherent CIC-enriched mammospheres. Immune-deficient mice bearing orthotopic SUM159 or 2LMP xenografts were injected i.v. with the targeted (225.28 or irrelevant isotype-matched control (F3-C25 mAbs, labeled with 99mTc, 125I, or 212Pb for in vivo imaging, biodistribution, or tumor growth inhibition studies. 212Pb-225.28 bound to adherent SUM159 and 2LMP cells and to CICs from SUM159 and 2LMP mammospheres with a mean affinity of 0.5 nM. Nearly ten times more binding sites per cell were present on SUM159 cells and CICs compared with 2LMP cells. 212Pb-225.28 was six to seven times more effective than 212Pb-F3-C25 at inhibiting SUM159 cell and CIC clonogenic survival (p < 0.05. Radiolabeled mAb 225.28 showed significantly higher uptake than radiolabeled mAb F3-C25 in SUM159 and 2LMP xenografts (p < 0.05, and the uptake of 212Pb-225.28 in TNBC xenografts was correlated with target epitope expression. 212Pb-225.28 caused dose

Structural analysis and anticoagulant activities of two sulfated polysaccharides from the sea cucumber Holothuria coluber.

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Yang, Wenjiao; Cai, Ying; Yin, Ronghua; Lin, Lisha; Li, Zhongkun; Wu, Mingyi; Zhao, Jinhua

2018-05-01

Sulfated polysaccharides such as fucosylated glycosaminoglycan and fucan sulfate from echinoderm possess complex chemical structure and various biological activities. The two sulfated polysaccharides were purified from the low-value sea cucumber Holothuria coluber. Their physicochemical properties and chemical structures were analyzed and characterized by chemical and instrumental methods. Structural analysis clarified that the sea cucumber fucosylated glycosaminoglycan contains a chondroitin sulfate-like backbone and fucosyl branches with four various sulfation patterns. The fucan sulfate with molecular weight of 64.6 kDa comprises a central core of regular α(1 → 4)-linked tetrasaccharide repeating units, each of which is linked by a 4-O-sulfated fucose residue. Anticoagulant assays indicated that these sulfated polysaccharides possessed strong APTT prolonging activities and intrinsic factor Xase inhibitory activities, both of which decreased with the reduction of their molecular weights. Our results expand knowledge on the structural types of sulfated polysaccharides from sea cucumbers and further illustrate their functionality. Copyright © 2018. Published by Elsevier B.V.

Eudragit ® FS 30 D polymeric films containing chondroitin sulfate as candidates for use in coating seeking modified delivery of drugs

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Camila Borges dos Reis

Full Text Available ABSTRACT Polymeric films associating different concentrations of Eudragit(r FS 30 D (EFS and chondroitin sulfate (CS were produced by casting for the development of a new target-specific site material. Formed films kept a final polymer mass of 4% (w/v in the following proportions: EFS 100:00 CS (control, EFS 95:05 CS, EFS 90:10 CS and EFS 80:20 CS. They were analyzed for physical and chemical characteristics using Fourier transform infrared spectroscopy (FTIR, scanning electron microscopy (SEM and Raman spectroscopy. Furthermore, they were characterized by their water vapor permeability and degree of hydration at different conditions simulating the gastrointestinal tract. No chemical interactions were observed between CS and EFS, suggesting only a physical interaction between them in the different combinations tested. The results suggest that EFS and CS, when combined, may form films that are candidates for coating processes seeking a modified drug delivery, especially due to the synergism between pH dependency and specific biodegradability properties by the colonic microbiota. EFS 90:10 CS proved to be the most suitable for this purpose considering hydration and permeability characteristics of different associations analyzed.

Efficacy of 2-Hydroxypropyl-β-cyclodextrin in Niemann-Pick Disease Type C Model Mice and Its Pharmacokinetic Analysis in a Patient with the Disease.

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Tanaka, Yuta; Yamada, Yusei; Ishitsuka, Yoichi; Matsuo, Muneaki; Shiraishi, Koki; Wada, Koki; Uchio, Yushiro; Kondo, Yuki; Takeo, Toru; Nakagata, Naomi; Higashi, Taishi; Motoyama, Keiichi; Arima, Hidetoshi; Mochinaga, Sakiko; Higaki, Katsumi; Ohno, Kousaku; Irie, Tetsumi

2015-01-01

Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disorder, is an inherited disease characterized by the accumulation of intracellular unesterified cholesterol. A solubilizing agent of lipophilic compounds, 2-hydroxypropyl-β-cyclodextrin (HPBCD), is an attractive drug candidate against NPC disease. However, establishment of the optimum dosage of HPBCD remains to be determined. In this study, we evaluated the effective dosage of HPBCD in NPC model (Npc1(-/-)) mice, and determined serum HPBCD concentrations. Subcutaneous injection of 1000-4000 mg/kg HPBCD improved the lifespan of Npc1(-/-) mice. In addition, liver injury and cholesterol sequestration were significantly prevented by 4000 mg/kg HPBCD in Npc1(-/-) mice. Serum HPBCD concentrations, when treated at the effective dosages (1000-4000 mg/kg), were approximately 1200-2500 µg/mL at 0.5 h after subcutaneous injection, and blood HPBCD concentrations were immediately eliminated in Npc1(-/-) mice. Furthermore, we examined serum HPBCD concentrations when treated at 40000 mg (approximately 2500 mg/kg) in a patient with NPC. We observed that the effective concentration in the in vivo study using Npc1(-/-) mice was similar to that in the patient. In the patient, systemic clearance and the volume of distribution of HPBCD were in accordance with the glomerular filtration rate and extracellular fluid volume, respectively. These results could provide useful information for developing the optimal dosage regimen for HPBCD therapy when administered intravenously to NPC patients.

Degree of Suppression of Mouse Myoblast Cell Line C₂C12 Differentiation Varies According to Chondroitin Sulfate Subtype.

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Warita, Katsuhiko; Oshima, Nana; Takeda-Okuda, Naoko; Tamura, Jun-Ichi; Hosaka, Yoshinao Z

2016-10-21

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C₂C 12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion.

Genetic Characterization of Movement Disorders and Dementias

Science.gov (United States)

2018-04-27

Ataxia; Dystonia; Parkinson's Disease; Amyotrophic Lateral Sclerosis; Corticobasal Degeneration; Multiple System Atrophy; Alzheimer's Disease; Lewy Body Dementia; Parkinson Disease-Dementia; Dentatorubral-pallidoluysian Atrophy; Creutzfeldt-Jakob Disease and Fatal Familial Insomnia; Fragile X-associated Tremor/Ataxia Syndrome; Krabbe's Disease; Niemann-Pick Disease, Type C; Neuronal Ceroid Lipofuscinosis

Protein complexes and cholesterol in the control of late endosomal dynamicsCholesterol and multi-protein complexes in the control of late endosomal dynamics

NARCIS (Netherlands)

Kant, Rik Henricus Nicolaas van der

2013-01-01

Late endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer’s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the

Microsphere-Based Scaffolds Carrying Opposing Gradients of Chondroitin Sulfate and Tricalcium Phosphate

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Vineet eGupta

2015-07-01

Full Text Available Extracellular matrix (ECM components such as chondroitin sulfate (CS and tricalcium phosphate (TCP serve as raw materials and thus spatial patterning of these raw materials may be leveraged to mimic the smooth transition of physical, chemical and mechanical properties at the bone-cartilage interface. We hypothesized that encapsulation of opposing gradients of these raw materials in high molecular weight poly(D,L-lactic-co-glycolic acid (PLGA microsphere-based scaffolds would enhance differentiation of rat bone marrow stromal cells (rBMSCs. The raw material encapsulation altered the microstructure of the microspheres and also influenced the cellular morphology that depended on the type of material encapsulated. Moreover, the mechanical properties of the raw material encapsulating microsphere-based scaffolds initially relied on the composition of the scaffolds and later on were primarily governed by the degradation of the polymer phase and newly synthesized extracellular matrix by the seeded cells. Furthermore, raw materials had a mitogenic effect on the seeded cells and led to increased glycosaminoglycan (GAG, collagen, and calcium content. Interestingly, the initial effects of raw material encapsulation on a per-cell basis might have been overshadowed by medium-regulated environment that appeared to favor osteogenesis. However, it is to be noted that in vivo, differentiation of the cells would be governed by the surrounding native environment. Thus, the results of this study demonstrated the potential of the raw materials in facilitating neo-tissue synthesis in microsphere-based scaffolds and perhaps in combination with bioactive signals, these raw materials may be able to achieve intricate cell differentiation profiles required for regenerating the osteochondral interface.

Molecular size is important for the safety and selective inhibition of intrinsic factor Xase for fucosylated chondroitin sulfate.

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Yan, Lufeng; Li, Junhui; Wang, Danli; Ding, Tian; Hu, Yaqin; Ye, Xingqian; Linhardt, Robert J; Chen, Shiguo

2017-12-15

Fucosylated chondroitin sulfate from sea cucumber Isostichopus badionotus (FCS-Ib) showed potent anticoagulant activities without selectivity. The present study focused on developing safe FCS-Ib oligomers showing selective inhibition of intrinsic factor Xase (anti-FXase) prepared through partial N-deacetylation-deaminative cleavage. The N-deacetylation degree was regulated by reaction time, controlling the resulting oligomer distribution. Structure analysis confirmed the selectivity of degradation, and 12 high purity fractions with trisaccharide-repeating units were separated. In vitro anticoagulant assays indicated a decrease in molecular weight (Mw) dramatically reduced activated partial thromboplastin time (APTT), thrombin time (TT), AT-dependent anti-FIIa and anti-FXa activities, while the oligomers retained potent anti-FXase activity until they fell below 3kDa. Meanwhile, human FXII activation and platelet aggregation were markedly reduced with decreasing Mw and were moderate when under 12.0kDa. Thus, fragments of 3-12.0kDa should be safe and effective as selective inhibitors of intrinsic tenase complex for application as clinical anticoagulants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electrospun Gelatin–Chondroitin Sulfate Scaffolds Loaded with Platelet Lysate Promote Immature Cardiomyocyte Proliferation

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Francesca Saporito

2018-02-01

Full Text Available The aim of the present work was the development of heart patches based on gelatin (G and chondroitin sulfate (CS to be used as implants to improve heart recovery after corrective surgery for critical congenital heart defects (CHD. Patches were prepared by means of electrospinning to obtain nanofibrous scaffolds and they were loaded with platelet lysate (PL as a source of growth factors to further enhance the repair process. Scaffolds were characterized for morphology and mechanical properties and for the capability to support in vitro adhesion and proliferation of dermal fibroblasts in order to assess the system’s general biocompatibility. Adhesion and proliferation of endothelial cells and cardiac cells (cardiomyocytes and cardiac fibroblasts from rat fetuses onto PL-loaded patches was evaluated. Patches presented good elasticity and high stiffness suitable for in vivo adaptation to heart contraction. CS improved adhesion and proliferation of dermal fibroblasts, as proof of their biocompatibility. Moreover, they enhanced the adhesion and proliferation of endothelial cells, a crucial mediator of cardiac repair. Cell adhesion and proliferation could be related to elastic properties, which could favor cell motility. The presence of platelet lysate and CS was crucial for the adhesion and proliferation of cardiac cells and, in particular, of cardiomyocytes: G/CS scaffold embedded with PL appeared to selectively promote proliferation in cardiomyocytes but not cardiac fibroblasts. In conclusion, G/CS scaffold seems to be a promising system to assist myocardial-repair processes in young patient, preserving cardiomyocyte viability and preventing cardiac fibroblast proliferation, likely reducing subsequent uncontrolled collagen deposition by fibroblasts following repair.

Identification of 58 novel mutations in Niemann-Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1.

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Park, Walter D; O'Brien, John F; Lundquist, Patrick A; Kraft, Daniel L; Vockley, Cate Walsh; Karnes, Pamela S; Patterson, Marc C; Snow, Karen

2003-10-01

The two known complementation groups of Niemann-Pick Type C disease, NPC1 and NPC2, result from non-allelic protein defects. Both the NPC1 and NPC2 (HE1) gene products are intimately involved in cholesterol and glycolipid trafficking and/or transport. We describe mutation analysis on samples from 143 unrelated affected NPC patients using conformation sensitive gel electrophoresis and DNA sequencing as the primary mutation screening methods for NPC1 and NPC2, respectively. These methods are robust, sensitive, and do not require any specialized laboratory equipment. Analyses identified two NPC1 mutations for 115 (80.4%) patients, one NPC1 mutation for 10 (7.0%) patients, two NPC2 mutations for five (3.5%) patients, one NPC2 mutation for one (0.7%) patient, and no mutations for 12 (8.4%) patients. Thus, mutations were identified on 251 of 286 (88%) disease alleles, including 121 different mutations (114 in NPC1 and seven in NPC2), 58 of which are previously unreported. The most common NPC1 mutation, I1061T, was detected on 18% of NPC alleles. Other NPC1 mutations were mostly private, missense mutations located throughout the gene with clustering in the cysteine-rich luminal domain. Correlation with biochemical data suggests classification of several mutations as severe and others as moderate or variable. The region between amino acids 1038 and 1253, which shares 35% identity with Patched 1, appears to be a hot spot for mutations. Additionally, a high percentage of mutations were located at amino acids identical to the NPC1 homolog, NPC1L1. Biochemical complementation analysis of cases negative for mutations revealed a high percentage of equivocal results where the complementation group appeared to be non-NPC1 and non-NPC2. This raises the possibilities of an additional NPC complementation group(s) or non-specificity of the biochemical testing for NPC. These caveats must be considered when offering mutation testing as a clinical service. Copyright 2003 Wiley-Liss, Inc.

Lysosomal pH-inducible supramolecular dissociation of polyrotaxanes possessing acid-labile N-triphenylmethyl end groups and their therapeutic potential for Niemann-Pick type C disease

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Tamura, Atsushi; Nishida, Kei; Yui, Nobuhiko

2016-01-01

Niemann-Pick type C (NPC) disease is characterized by the accumulation of cholesterol in lysosomes. We have previously reported that biocleavable polyrotaxanes (PRXs) composed of β-cyclodextrins (β-CDs) threaded onto a linear polymer capped with bulky stopper molecules via intracellularly cleavable linkers show remarkable cholesterol reducing effects in NPC disease patient-derived fibroblasts owing to the stimuli-responsive intracellular dissociation of PRXs and subsequent β-CD release from the PRXs. Herein, we describe a series of novel acid-labile 2-(2-hydroxyethoxy)ethyl group-modified PRXs (HEE-PRXs) bearing terminal N-triphenylmethyl (N-Trt) groups as a cleavable component for the treatment of NPC disease. The N-Trt end groups of the HEE-PRXs underwent acidic pH-induced cleavage and led to the dissociation of their supramolecular structure. A kinetic study revealed that the number of HEE groups on the PRX did not affect the cleavage kinetics of the N-Trt end groups of the HEE-PRXs. The effect of the number of HEE groups of the HEE-PRXs, which was modified to impart water solubility to the PRXs, on cellular internalization efficiency, lysosomal localization efficiency, and cholesterol reduction ability in NPC disease-derived fibroblasts (NPC1 fibroblasts) was also investigated. The cellular uptake and lysosomal localization efficiency were almost equivalent for HEE-PRXs with different numbers of HEE groups. However, the cholesterol reducing ability of the HEE-PRXs in NPC1 fibroblasts was affected by the number of HEE groups, and HEE-PRXs with a high number of HEE groups were unable to reduce lysosomal cholesterol accumulation. This deficiency is most likely due to the cholesterol-solubilizing ability of HEE-modified β-CDs released from the HEE-PRXs. We conclude that the N-Trt group acts as a cleavable component to induce the lysosomal dissociation of HEE-PRXs, and acid-labile HEE-PRXs with an optimal number of HEE groups (4.1 to 5.4 HEE groups per single �

Fabrication of Chitin/Poly(butylene succinate/Chondroitin Sulfate Nanoparticles Ternary Composite Hydrogel Scaffold for Skin Tissue Engineering

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S. Deepthi

2014-12-01

Full Text Available Skin loss is one of the oldest and still not totally resolved problems in the medical field. Since spontaneous healing of the dermal defects would not occur, the regeneration of full thickness of skin requires skin substitutes. Tissue engineering constructs would provide a three dimensional matrix for the reconstruction of skin tissue and the repair of damage. The aim of the present work is to develop a chitin based scaffold, by blending it with poly(butylene succinate (PBS, an aliphatic, biodegradable and biocompatible synthetic polymer with excellent mechanical properties. The presence of chondroitin sulfate nanoparticles (CSnp in the scaffold would favor cell adhesion. A chitin/PBS/CSnp composite hydrogel scaffold was developed and characterized by SEM (Scanning Electron Microscope, FTIR (Fourier Transform Infrared Spectroscopy, and swelling ratio of scaffolds were analyzed. The scaffolds were evaluated for the suitability for skin tissue engineering application by cytotoxicity, cell attachment, and cell proliferation studies using human dermal fibroblasts (HDF. The cytotoxicity and cell proliferation studies using HDF confirm the suitability of the scaffold for skin regeneration. In short, these results show promising applicability of the developed chitin/PBS/CSnps ternary composite hydrogel scaffolds for skin tissue regeneration.

Effect of Bioactive Materials Modified with Chondroitin Sulfate on Human MSC =

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De La Torre Torres, Jessica Elizabeth

In this project chondroitin sulfate (CS) and growth factors were studied for their effect on hMSC in biomaterials. First, the effect of these biomolecules was tested in solution. Then, two kinds of biomaterials were created: bioactive surfaces for enhancing bioactivity of implantable devices and bioactive hydrogels which can be used as 3D scaffolds for cell encapsulation and delivery. A pro-survival effect of the growth factors studied in this project (epidermal growth factor, vascular endothelial growth factor and fibroblast growth factor) was not observed when tested in solution, therefore the project further focused on CS effect only. Interestingly, CS did not affect cell growth in media containing serum, while inducing cell detachment from substrate in serum free conditions. For the bioactive surfaces construction, CS was grafted to either an amine-rich plasmapolymerized coating created on polyethylene terephthalate (PET) films (further referred as LP) or to commercial cell culture plates functionalized with amino groups. The bioactive surfaces were characterized by different techniques such as contact angle, atomic force microscopy, Orange II dye and Toluidine Blue O dye colorimetric assays (for amino group and CS quantification respectively) and finally, cell culture experiments (adhesion, growth and survival). Results confirmed the presence of CS grafted on both substrates. Commercial amine plates grafted almost five times more CS compared to LP. This rendered the surface antifouling for proteins and cells as confirmed by protein adsorption and cell culture assays. Cell culture assays on bioactive surfaces based on LP demonstrated improved cell adhesion and growth when compared to tissue culture plates or bare PET films in serum containing conditions. Chitosan based hydrogels containing CS at a concentration of 500 mug/ml resulted in a cohesive hydrogel which supported hMSC viability up to 7 days. However increasing CS concentration to high level such as

Structure of the DBL3x domain of pregnancy-associated malaria protein VAR2CSA complexed with chondroitin sulfate A

Energy Technology Data Exchange (ETDEWEB)

Singh, K.; Gittis, A.G.; Nguyen, P.; Gowda, D.C.; Miller, L.H.; Garboczi, D.N. (NIH); (Penn)

2008-09-19

Plasmodium falciparum-infected erythrocytes bind to chondroitin sulfate A (CSA) in the placenta via the VAR2CSA protein, a member of the P. falciparum erythrocyte membrane protein-1 family, leading to life-threatening malaria in pregnant women with severe effects on their fetuses and newborns. Here we describe the structure of the CSA binding DBL3x domain, a Duffy binding-like (DBL) domain of VAR2CSA. By forming a complex of DBL3x with CSA oligosaccharides and determining its structure, we have identified the CSA binding site to be a cluster of conserved positively charged residues on subdomain 2 and subdomain 3. Mutation or chemical modification of lysine residues at the site markedly diminished CSA binding to DBL3x. The location of the CSA binding site is an important step forward in the molecular understanding of pregnancy-associated malaria and offers a new target for vaccine development.

Chondroitin-6-sulfate attenuates inflammatory responses in murine macrophages via suppression of NF-κB nuclear translocation.

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Tan, Guak-Kim; Tabata, Yasuhiko

2014-06-01

Inflammation is a host protective response to noxious stimuli, and excessive production of pro-inflammatory mediators by macrophages (mφ) can lead to numerous pathological conditions. In this study, immunomodulatory effects of immobilized and soluble glycosaminoglycans (GAGs) on mouse-bone-marrow-derived mφ were compared by measuring nitric oxide (NO). We demonstrate here that all GAGs studied except for heparin were able to modulate interferon-γ/lipopolysaccharide (IFN-γ/LPS)-induced NO release by mφ to varying extents after 24h of incubation. In particular, the modulatory activities of soluble chondroitin-6-sulfate (C6S), hyaluronic acid and heparan sulfate altered markedly after covalent immobilization. Of these, soluble C6S exhibited the strongest NO inhibitory activity, and the inhibition was dose- and time-dependent. Moreover, C6S significantly reduced pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α production by IFN-γ/LPS- or LPS-activated mφ. Specifically, the C6S-mediated suppression of mφ pro-inflammatory phenotype was accompanied by an increase in the IL-10 level, suggesting a possible switch towards anti-inflammatory/wound healing M2 state. In addition, the highest magnitude of inhibitory effects was obtained when cells were pre-treated with C6S prior to IFN-γ/LPS or LPS challenge, suggesting an additional role for C6S in protection against microbial infection. Further investigations reveal that the anti-inflammatory effects of C6S on activated mφ may be ascribed at least in part to suppression of NF-κB nuclear translocation. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Bonding and fusion of meniscus fibrocartilage using a novel chondroitin sulfate bone marrow tissue adhesive.

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Simson, Jacob A; Strehin, Iossif A; Allen, Brian W; Elisseeff, Jennifer H

2013-08-01

The weak intrinsic meniscus healing response and technical challenges associated with meniscus repair contribute to a high rate of repair failures and meniscectomies. Given this limited healing response, the development of biologically active adjuncts to meniscal repair may hold the key to improving meniscal repair success rates. This study demonstrates the development of a bone marrow (BM) adhesive that binds, stabilizes, and stimulates fusion at the interface of meniscus tissues. Hydrogels containing several chondroitin sulfate (CS) adhesive levels (30, 50, and 70 mg/mL) and BM levels (30%, 50%, and 70%) were formed to investigate the effects of these components on hydrogel mechanics, bovine meniscal fibrochondrocyte viability, proliferation, matrix production, and migration ability in vitro. The BM content positively and significantly affected fibrochondrocyte viability, proliferation, and migration, while the CS content positively and significantly affected adhesive strength (ranged from 60±17 kPa to 335±88 kPa) and matrix production. Selected material formulations were translated to a subcutaneous model of meniscal fusion using adhered bovine meniscus explants implanted in athymic rats and evaluated over a 3-month time course. Fusion of adhered meniscus occurred in only the material containing the highest BM content. The technology can serve to mechanically stabilize the tissue repair interface and stimulate tissue regeneration across the injury site.

Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture

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Jingyu Jin

2018-01-01

Full Text Available As one major component of extracellular matrix (ECM in the central nervous system, chondroitin sulfate proteoglycans (CSPGs have long been known as inhibitors enriched in the glial scar that prevent axon regeneration after injury. Although many studies have shown that CSPGs inhibited neurite outgrowth in vitro using different types of neurons, the mechanism by which CSPGs inhibit axonal growth remains poorly understood. Using cerebellar granule neuron (CGN culture, in this study, we evaluated the effects of different concentrations of both immobilized and soluble CSPGs on neuronal growth, including cell adhesion, spreading and neurite growth. Neurite length decreased while CSPGs concentration arised, meanwhile, a decrease in cell density accompanied by an increase in cell aggregates formation was observed. Soluble CSPGs also showed an inhibition on neurite outgrowth, but it required a higher concentration to induce cell aggregates formation than coated CSPGs. We also found that growth cone size was significantly reduced on CSPGs and neuronal cell spreading was restrained by CSPGs, attributing to an inhibition on lamellipodial extension. The effect of CSPGs on neuron adhesion was further evidenced by interference reflection microscopy (IRM which directly demonstrated that both CGNs and cerebral cortical neurons were more loosely adherent to a CSPG substrate. These data demonstrate that CSPGs have an effect on cell adhesion and spreading in addition to neurite outgrowth.

Chondroitin sulfate immobilization at the surface of electrospun nanofiber meshes for cartilage tissue regeneration approaches

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Piai, Juliana Francis; da Silva, Marta Alves; Martins, Albino; Torres, Ana Bela; Faria, Susana; Reis, Rui L.; Muniz, Edvani Curti; Neves, Nuno M.

2017-05-01

Aiming at improving the biocompatibility of biomaterial scaffolds, surface modification presents a way to preserve their mechanical properties and to improve the surface bioactivity. In this work, chondroitin sulfate (CS) was immobilized at the surface of electrospun poly(caprolactone) nanofiber meshes (PCL NFMs), previously functionalized by UV/O3 exposure and aminolysis. Contact angle, SEM, optical profilometry, FTIR, X-ray photoelectron spectroscopy techniques confirmed the success of CS-immobilization in PCL NFMs. Furthermore, CS-immobilized PCL NFMs showed lower roughness and higher hydrophilicity than the samples without CS. Human articular chondrocytes (hACs) were cultured on electrospun PCL NFMs with or without CS immobilization. It was observed that hACs proliferated through the entire time course of the experiment in both types of nanofibrous scaffolds, as well as for the production of glycosaminoglycans. Quantitative-PCR results demonstrated over-expression of cartilage-related genes such as Aggrecan, Collagen type II, COMP and Sox9 on both types of nanofibrous scaffolds. Morphological observations from SEM and LSCM revealed that hACs maintained their characteristic round shape and cellular agglomeration exclusively on PCL NFMs with CS immobilization. In conclusion, CS immobilization at the surface of PCL NFMs was achieved successfully and provides a valid platform enabling further surface functionalization methods in scaffolds to be developed for cartilage tissue engineering.

2-Amino-4-hydroxyethylaminoanisole sulfate

DEFF Research Database (Denmark)

Madsen, Jakob T; Andersen, Klaus E

2016-01-01

positive patch test reactions to the coupler 2-amino-4-hydroxyethylaminoanisole sulfate 2% pet. from 2005 to 2014. METHODS: Patch test results from the Allergen Bank database for eczema patients patch tested with 2-amino-4-hydroxyethylaminoanisole sulfate 2% pet. from 2005 to 2014 were reviewed. RESULTS......: A total of 902 dermatitis patients (154 from the dermatology department and 748 from 65 practices) were patch tested with amino-4-hydroxyethylaminoanisole sulfate 2% pet. from 2005 to 2014. Thirteen (1.4%) patients had a positive patch test reaction. Our results do not indicate irritant reactions....... CONCLUSIONS: 2-Amino-4-hydroxyethylaminoanisole sulfate is a new but rare contact allergen....

Transforming growth factor β-induced expression of chondroitin sulfate proteoglycans is mediated through non-Smad signaling pathways.

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Jahan, Naima; Hannila, Sari S

2015-01-01

The expression of chondroitin sulfate proteoglycans (CSPGs) by reactive astrocytes is a major factor contributing to glial scarring and regenerative failure after spinal cord injury, but the molecular mechanisms underlying CSPG expression remain largely undefined. One contributing factor is transforming growth factor β (TGFβ), which is upregulated after injury and has been shown to induce expression of CSPGs in vitro. TGFβ typically mediates its effects through the Smad2/3 signaling pathway, and it has been suggested that this pathway is responsible for CSPG expression. However, there is evidence that TGFβ can also activate non-Smad signaling pathways. In this study, we report that TGFβ-induced expression of three different CSPGs--neurocan, brevican, and aggrecan--is mediated through non-Smad signaling pathways. We observed significant increases in TGFβ-induced expression of neurocan, brevican, and aggrecan following siRNA knockdown of Smad2 or Smad4, which indicates that Smad signaling is not required for the expression of these CSPGs. In addition, we show that neurocan, aggrecan, and brevican levels are significantly reduced when TGFβ is administered in the presence of either the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin, but not the MEK1/2 inhibitor U0126. This suggests that TGFβ mediates this effect through non-Smad-dependent activation of the PI3K-Akt-mTOR signaling pathway, and targeting this pathway may therefore be an effective means of reducing CSPG expression in the injured CNS. Copyright © 2014 Elsevier Inc. All rights reserved.

Sulfated glycosaminoglycans in human vocal fold lamina propria

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Sung Woo Park

Full Text Available Abstract Introduction: The distribution, concentration and function of glycosaminoglycans in the various vocal fold tissues are still unclear. Objective: To evaluate the distribution and concentration of sulfated glycosaminoglycans in different layers of the human vocal fold according to gender and age. Methods: We used 11 vocal folds obtained from cadavers (7 men and 4 women with no laryngeal lesion, less than 12 h after death, and aged between 35 and 98 years. The folds underwent glycosaminoglycans extraction from the cover and ligament, and post-electrophoresis analysis. Data were compared according to the layer, age and gender. Results: The concentration of dermatan sulfate was significantly higher in all layers. No differences were observed in the total concentrations of glycosaminoglycans in layers studied according to gender. It is significantly lower in the cover of individuals aged below 60 years. Conclusion: Dermatan sulfate, chondroitin sulfate, and heparan sulfate were observed in the human vocal folds cover and ligament of both genders, with the concentration of dermatan sulfate being significantly higher in all layers. Glycosaminoglycans concentration on the cover is significantly lower in individuals below 60 years compared with elderly.

Protein replacement therapy partially corrects the cholesterol-storage phenotype in a mouse model of Niemann-Pick type C2 disease.

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Gitte Krogh Nielsen

Full Text Available Niemann-Pick type C2 (NPC2 disease is a fatal autosomal recessive neurovisceral degenerative disorder characterized by late endosomal-lysosomal sequestration of low-density lipoprotein derived cholesterol. The breach in intracellular cholesterol homeostasis is caused by deficiency of functional NPC2, a soluble sterol binding protein targeted to the lysosomes by binding the mannose-6-phosphate receptor. As currently there is no effective treatment for the disorder, we have investigated the efficacy of NPC2 replacement therapy in a murine gene-trap model of NPC2-disease generated on the 129P2/OlaHsd genetic background. NPC2 was purified from bovine milk and its functional competence assured in NPC2-deficient fibroblasts using the specific cholesterol fluorescent probe filipin. For evaluation of phenotype correction in vivo, three-week-old NPC2(-/- mice received two weekly intravenous injections of 5 mg/kg NPC2 until trial termination 66 days later. Whereas the saline treated NPC2(-/- mice exhibited massive visceral cholesterol storage as compared to their wild-type littermates, administration of NPC2 caused a marked reduction in cholesterol build up. The histological findings, indicating an amelioration of the disease pathology in liver, spleen, and lungs, corroborated the biochemical results. Little or no difference in the overall cholesterol levels was observed in the kidneys, blood, cerebral cortex and hippocampus when comparing NPC2(-/- and wild type mice. However, cerebellum cholesterol was increased about two fold in NPC2(-/- mice compared with wild-type littermates. Weight gain performance was slightly improved as a result of the NPC2 treatment but significant motor coordination deficits were still observed. Accordingly, ultrastructural cerebellar abnormalities were detected in both saline treated and NPC2 treated NPC2(-/- animals 87 days post partum. Our data indicate that protein replacement may be a beneficial therapeutic approach in the

Pre-symptomatic activation of antioxidant responses and alterations in glucose and pyruvate metabolism in Niemann-Pick Type C1-deficient murine brain.

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Barry E Kennedy

Full Text Available Niemann-Pick Type C (NPC disease is an autosomal recessive neurodegenerative disorder caused in most cases by mutations in the NPC1 gene. NPC1-deficiency is characterized by late endosomal accumulation of cholesterol, impaired cholesterol homeostasis, and a broad range of other cellular abnormalities. Although neuronal abnormalities and glial activation are observed in nearly all areas of the brain, the most severe consequence of NPC1-deficiency is a near complete loss of Purkinje neurons in the cerebellum. The link between cholesterol trafficking and NPC pathogenesis is not yet clear; however, increased oxidative stress in symptomatic NPC disease, increases in mitochondrial cholesterol, and alterations in autophagy/mitophagy suggest that mitochondria play a role in NPC disease pathology. Alterations in mitochondrial function affect energy and neurotransmitter metabolism, and are particularly harmful to the central nervous system. To investigate early metabolic alterations that could affect NPC disease progression, we performed metabolomics analyses of different brain regions from age-matched wildtype and Npc1 (-/- mice at pre-symptomatic, early symptomatic and late stage disease by (1H-NMR spectroscopy. Metabolic profiling revealed markedly increased lactate and decreased acetate/acetyl-CoA levels in Npc1 (-/- cerebellum and cerebral cortex at all ages. Protein and gene expression analyses indicated a pre-symptomatic deficiency in the oxidative decarboxylation of pyruvate to acetyl-CoA, and an upregulation of glycolytic gene expression at the early symptomatic stage. We also observed a pre-symptomatic increase in several indicators of oxidative stress and antioxidant response systems in Npc1 (-/- cerebellum. Our findings suggest that energy metabolism and oxidative stress may present additional therapeutic targets in NPC disease, especially if intervention can be started at an early stage of the disease.

Seven novel mutations of the SMPD1 gene in four Chinese patients with Niemann-Pick disease type A and prenatal diagnosis for four fetuses.

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Ding, Yuan; Li, Xiyuan; Liu, Yupeng; Hua, Ying; Song, Jinqing; Wang, Liwen; Li, Mengqiu; Qin, Yaping; Yang, Yanling

2016-04-01

Niemann-Pick disease type A (NPD-A) is a rare autosomal recessive lysosomal storage disorder caused by acid sphingomyelinase deficiency. Only a few cases have been documented in mainland China, and prenatal diagnosis has not been performed to date. In this study, the clinical and laboratory features of four Chinese patients with early-onset NPD-A were summarized. Four patients with NPD-A were the firstborns of non-consanguineous parents from four unrelated Chinese families. Bone marrow analysis, acid sphingomyelinase assay and genetic studies were performed. SMPD1 gene studies on amniocytes were performed for the prenatal diagnosis of four fetuses from three families. Four patients were admitted at the age of 1-10 months due to jaundice, hepatosplenomegaly and psychomotor retardation. Liver histopathological analysis revealed glucolipid accumulation. Massive foamy histiocytes were found in the bone marrow. Acid sphingomyelinase activities of peripheral blood leukocytes were significantly decreased (4.05-21.9 nmol/h/mg protein, normal range 216.1-950.9 nmol/h/mg protein). Seven novel mutations (c.518-519insT, c.562_563insC, c.792Gdel, c.949G>A, c.1487_1499delACCGTGTGTACCA, c.1495T>C and c.1670T>C) of the SMPD1 gene were identified in four patients. Only one fetus had two mutations of the SMPD1 gene of amniocytes. The results suggested that the fetus was affected by NPD-A. The mother chose artificial abortion. The other three fetuses were not affected by NPD-A. No mutation of the SMPD1 gene was detected in the cultured amniocytes from the mothers. Postnatal genetic analysis and normal development of the three infants confirmed the prenatal diagnosis. Seven novel mutations associated with NPD-A were identified in the Chinese population. Prenatal diagnosis for four fetuses of three families was successfully performed by amniocyte gene analysis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Layer-by-layer assembly of type I collagen and chondroitin sulfate on aminolyzed PU for potential cartilage tissue engineering application

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He, Xianyun; Wang, Yingjun; Wu, Gang

2012-10-01

In this paper, a two-step method was used to synthesize a biodegradable polyurethane (PU) composed of L-lysine ethyl ester diisocyanate (LDI), poly(ɛ-caprolactone) diols (PCL-diol) and 1,4:3,6-dianhydro-D-sorbitol (isosorbide). Amino groups were introduced onto the surface of the PU membrane by an amination reacting with 1,3-propanediamine to produce polycationic substratum. And then, type I collagen (Col) and chondroitin sulfate (CS) were deposited alternately on the polycationic substratum through layer-by-layer (LBL) assembly technology. The FTIR and 1H NMR results showed that the polyurethane was successfully synthesized. Rhodamine B isothiocyanate (RBITC) fluorescence spectrum indicated that amino groups were successfully introduced onto the PU surface. The results of quartz-crystal microbalance (QCM) and RBITC-Col fluorescence spectroscopy monitoring the LBL assemble process presented that the Col/CS deposited alternately on the PU surface. X-ray photoelectron spectroscopy (XPS) results displayed that the CS deposited on the PU surface as well. The surface of the assembled PU became even smoother observed from the surface morphology by atomic force microscopy (AFM) imaging. The hydrophilicity of the PU membrane was greatly enhanced though the modification of LBL assembly. The PU modified with the adsorption of Col/CS may be a potential application for cartilage tissue engineering due to its created mimicking chondrogenic environment.

Burkitt lymphoma express oncofetal Chondroitin Sulfate without being a reservoir for placental malaria sequestration

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Agerbæk, Mette Ø.; Pereira, Marina A.; Clausen, Thomas M.; Pehrson, Caroline; Oo, Htoo Zarni; Spliid, Charlotte; Rich, Jamie R.; Fung, Vincent; Nkrumah, Francis; Neequaye, Janet; Biggar, Robert J.; Reynolds, Steven J.; Tosato, Giovanna; Pullarkat, Sheeja T.; Ayers, Leona W.; Theander, Thor G.; Daugaard, Mads; Bhatia, Kishor; Nielsen, Morten A.; Mbulaiteye, Sam M.; Salanti, Ali

2016-01-01

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. OfCS is absent from other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor-site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue, encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy. PMID:27997697

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration.

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Agerbaek, Mette Ø; Pereira, Marina A; Clausen, Thomas M; Pehrson, Caroline; Oo, Htoo Zarni; Spliid, Charlotte; Rich, Jamie R; Fung, Vincent; Nkrumah, Francis; Neequaye, Janet; Biggar, Robert J; Reynolds, Steven J; Tosato, Giovanna; Pullarkat, Sheeja T; Ayers, Leona W; Theander, Thor G; Daugaard, Mads; Bhatia, Kishor; Nielsen, Morten A; Mbulaiteye, Sam M; Salanti, Ali

2017-04-01

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy. © 2016 UICC.

Gamma ray-induced synthesis of hyaluronic acid/chondroitin sulfate-based hydrogels for biomedical applications

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Zhao, Linlin; Gwon, Hui-Jeong; Lim, Youn-Mook; Nho, Young-Chang; Kim, So Yeon

2015-01-01

Hyaluronic acid (HA)/chondroitin sulfate (CS)/poly(acrylic acid) (PAAc) hydrogel systems were synthesized by gamma-ray irradiation without the use of additional initiators or crosslinking agents to achieve a biocompatible hydrogel system for skin tissue engineering. HA and CS derivatives with polymerizable residues were synthesized. Then, the hydrogels composed of glycosaminoglycans, HA, CS, and a synthetic ionic polymer, PAAc, were prepared using gamma-ray irradiation through simultaneous free radical copolymerization and crosslinking. The physicochemical properties of the HA/CS/PAAc hydrogels having various compositions were investigated to evaluate their feasibility as artificial skin substitutes. The gel fractions of the HA/CS/PAAc hydrogels increased in absorbed doses up to 15 kGy, and they exhibited 91-93% gel fractions under 15 kGy radiation. All of the HA/CS/PAAc hydrogels exhibited relatively high water contents of over 90% and reached an equilibrium swelling state within 24 h. The enzymatic degradation kinetics of the HA/CS/PAAc hydrogels depended on both the concentration of the hyaluronidase solution and the ratio of HA/CS/PAAc. The in vitro drug release profiles of the HA/CS/PAAc hydrogels were significantly influenced by the interaction between the ionic groups in the hydrogels and the ionic drug molecules as well as the swelling of the hydrogels. From the cytotoxicity results of human keratinocyte (HaCaT) cells cultured with extracts of the HA/CS/PAAc hydrogels, all of the HA/CS/PAAc hydrogel samples tested showed relatively high cell viabilities of more than 82%, and did not induce any significant adverse effects on cell viability.

Biocompatibility Assessment of Novel Collagen-Sericin Scaffolds Improved with Hyaluronic Acid and Chondroitin Sulfate for Cartilage Regeneration

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Sorina Dinescu

2013-01-01

Full Text Available Cartilage tissue engineering (CTE applications are focused towards the use of implantable biohybrids consisting of biodegradable scaffolds combined with in vitro cultured cells. Hyaluronic acid (HA and chondroitin sulfate (CS were identified as the most potent prochondrogenic factors used to design new biomaterials for CTE, while human adipose-derived stem cells (ASCs were proved to display high chondrogenic potential. In this context, our aim was not only to build novel 3D porous scaffolds based on natural compounds but also to evaluate their in vitro biological performances. Therefore, for prospective CTE, collagen-sericin (Coll-SS scaffolds improved with HA (5% or 10% and CS (5% or 10% were used as temporary physical supports for ASCs and were analyzed in terms of structural, thermal, morphological, and swelling properties and cytotoxic potential. To complete biocompatibility data, ASCs viability and proliferation potential were also assessed. Our studies revealed that Coll-SS hydrogels improved with 10% HA and 5% CS displayed the best biological performances in terms of cell viability, proliferation, morphology, and distribution. Thus, further work will address a novel 3D system including both HA 10% and CS 5% glycoproteins, which will probably be exposed to prochondrogenic conditions in order to assess its potential use in CTE applications.

The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E.

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Harris, Edward N; Weigel, Paul H

2008-08-01

The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341-17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. (125)I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.

Chondroitin Sulfate Immobilized on a Biomimetic Scaffold Modulates Inflammation While Driving Chondrogenesis.

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Corradetti, Bruna; Taraballi, Francesca; Minardi, Silvia; Van Eps, Jeffrey; Cabrera, Fernando; Francis, Lewis W; Gazze, Salvatore A; Ferrari, Mauro; Weiner, Bradley K; Tasciotti, Ennio

2016-05-01

Costs associated with degenerative inflammatory conditions of articular cartilage are exponentially increasing in the aging population, and evidence shows a strong clinical need for innovative therapies. Stem cell-based therapies represent a promising strategy for the treatment of innumerable diseases. Their regenerative potential is undeniable, and it has been widely exploited in many tissue-engineering approaches, especially for bone and cartilage repair. Their immune-modulatory capacities in particular make stem cell-based therapeutics an attractive option for treating inflammatory diseases. However, because of their great plasticity, mesenchymal stem cells (MSCs) are susceptible to different external factors. Biomaterials capable of concurrently providing physical support to cells while acting as synthetic extracellular matrix have been established as a valuable strategy in cartilage repair. Here we propose a chondroitin sulfate-based biomimetic scaffold that recapitulates the physicochemical features of the chondrogenic niche and retains MSC immunosuppressive potential in vitro, either in response to a proinflammatory cytokine or in the presence of stimulated peripheral blood mononuclear cells. In both cases, a significant increase in the production of molecules associated with immunosuppression (nitric oxide and prostaglandins), as well as in the expression of their inducible enzymes (iNos, Pges, Cox-2, and Tgf-β). When implanted subcutaneously in rats, our scaffold revealed a reduced infiltration of leukocytes at 24 hours, which correlated with a greater upregulation of genes involved in inflammatory cell apoptotic processes. In support of its effective use in tissue-engineering applications of cartilage repair, the potential of the proposed platform to drive chondrogenic and osteogenic differentiation of MSC was also proven. Recently, increasing clinical evidence has highlighted the important role of proinflammatory mediators and infiltrating inflammatory

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

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von Einem, Bjoern; Weber, Petra; Wagner, Michael; Malnar, Martina; Kosicek, Marko; Hecimovic, Silva; Arnim, Christine A F von; Schneckenburger, Herbert

2012-11-26

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

Is intravesical instillation of hyaluronic acid and chondroitin sulfate useful in preventing recurrent bacterial cystitis? A multicenter case control analysis.

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Gugliotta, Giorgio; Calagna, Gloria; Adile, Giorgio; Polito, Salvatore; Saitta, Salvatore; Speciale, Patrizia; Palomba, Stefano; Perino, Antonino; Granese, Roberta; Adile, Biagio

2015-10-01

Urinary tract infections (UTIs) are common in the female population and, over a lifetime, about half of women have at least one episode of UTI requiring antibiotic therapy. The aim of the current study was to compare two different strategies for preventing recurrent bacterial cystitis: intravesical instillation of hyaluronic acid (HA) plus chondroitin sulfate (CS), and antibiotic prophylaxis with sulfamethoxazole plus trimethoprim. This was a retrospective review of two different cohorts of women affected by recurrent bacterial cystitis. Cases (experimental group) were women who received intravesical instillations of a sterile solution of high concentration of HA + CS in 50 mL water with calcium chloride every week during the 1(st) month and then once monthly for 4 months. The control group included women who received traditional therapy for recurrent cystitis based on daily antibiotic prophylaxis using sulfamethoxazole 200 mg plus trimethoprim 40 mg for 6 weeks. Ninety-eight and 76 patients were treated with experimental and control treatments, respectively. At 12 months after treatment, 69 and 109 UTIs were detected in the experimental and control groups, respectively. The proportion of patients free from UTIs was significantly higher in the experimental than in the control group (36.7% vs. 21.0%; p = 0.03). Experimental treatment was well tolerated and none of the patients stopped it. The intravesical instillation of HA + CS is more effective than long-term antibiotic prophylaxis for preventing recurrent bacterial cystitis. Copyright © 2015. Published by Elsevier B.V.

Collageneous matrix coatings on titanium implants modified with decorin and chondroitin sulfate: characterization and influence on osteoblastic cells.

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Bierbaum, Susanne; Douglas, Timothy; Hanke, Thomas; Scharnweber, Dieter; Tippelt, Sonja; Monsees, Thomas K; Funk, Richard H W; Worch, Hartmut

2006-06-01

Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM), utilizing the natural self-assembly potential of collagen in combination with further ECM components in close analogy to the situation in vivo. Aiming at load-bearing applications in bone contact, collagen type I in combination with the proteoglycan decorin and the glycosaminoglycan chondroitin sulfate (CS) was used; fibrillogenesis, fibril morphology, and adsorption of differently composed fibrils onto titanium were assessed. Both decorin and CS could be integrated into the fibrils during fibrillogenesis, the amount bound respectively desorbed depending on the ionic strength of fibrillogenesis buffer. Including decorin always resulted in a significant decrease of fibril diameter, CS in only a slight decrease or even increase, depending on the collagen preparation used. No significant changes in adsorption to titanium could be detected. Osteoblastic cells showed different reactions for cytoskeletal arrangement and osteopontin expression depending on the composition of the ECM, with CS enhancing the osteoblast phenotype.

Immobilized Lentivirus Vector on Chondroitin Sulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold for Tissue-Engineered Ligament-Bone Junction

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Liguo Sun

2014-01-01

Full Text Available The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor-β3 (TGF-β3 gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-β3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.

Spectral study of interaction between chondroitin sulfate and nanoparticles and its application in quantitative analysis

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Ma, Yi; Wei, Maojie; Zhang, Xiao; Zhao, Ting; Liu, Xiumei; Zhou, Guanglian

2016-01-01

In this work, the interaction between chondroitin sulfate (CS) and gold nanoparticles (GNPs) and silver nanoparticles (SNPs) was characterized for the first time. Plasma resonance scattering (PRS) and plasma resonance absorption (PRA) were used to investigate the characteristics of their spectrum. The results suggested that the CS with negative charge could interact with metal nanoparticles with negative charge and the adsorption of CS on the surface of SNPs was more regular than that of GNPs. The resonance scattering spectra also further confirmed the interaction between CS and SNPs. A new method for detection of CS based on the interaction was developed. CS concentrations in the range of 0.02-3.5 μg/mL were proportional to the decreases of absorbance of SNPs. Compared with other reported methods, the proposed method is simple and workable without complex process, high consumption and expensive equipments. The developed method was applied to the determination of the CS contents from different biological origins and the results were compared with those obtained by the method of Chinese Pharmacopeia. The effects of matrix in plasma and other glycosaminoglycans on the determination of CS were also investigated. The results showed that a small quantity of blood plasma had no effect on the determination of CS and when the concentration ratio of CS to heparin was more than 10:1, the influence of heparin on the detection of CS could be ignored. This work gave a specific research direction for the detection of CS in the presence of metal nanoparticles.

Determination of thermodynamic parameters for complexation of calcium and magnesium with chondroitin sulfate isomers using isothermal titration calorimetry: Implications for calcium kidney-stone research

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Rodgers, Allen L.; Jackson, Graham E.

2017-04-01

Chondroitin sulfate (CS) occurs in human urine. It has several potential binding sites for calcium and as such may play an inhibitory role in calcium oxalate and calcium phosphate (kidney stone disease by reducing the supersaturation (SS) and crystallization of these salts. Urinary magnesium is also a role player in determining speciation in stone forming processes. This study was undertaken to determine the thermodynamic parameters for binding of the disaccharide unit of two different CS isomers with calcium and magnesium. These included the binding constant K. Experiments were performed using an isothermal titration calorimeter (ITC) at 3 different pH levels in the physiological range in human urine. Data showed that interactions between the CS isomers and calcium and magnesium occur via one binding site, thought to be sulfate, and that log K values are 1.17-1.93 and 1.77-1.80 for these two metals respectively. Binding was significantly stronger in Mg-CS than in Ca-CS complexes and was found to be dependent on pH in the latter but not in the former. Furthermore, binding in Ca-CS complexes was dependent on the location of the sulfate binding site. This was not the case in the Mg-CS complexes. Interactions were shown to be entropy driven and enthalpy unfavourable. These findings can be used in computational modeling studies to predict the effects of the calcium and magnesium CS complexes on the speciation of calcium and the SS of calcium salts in real urine samples.

Chondroitin sulfate microparticles modulate transforming growth factor-β1-induced chondrogenesis of human mesenchymal stem cell spheroids.

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Goude, Melissa C; McDevitt, Todd C; Temenoff, Johnna S

2014-01-01

Mesenchymal stem cells (MSCs) have been previously explored as a part of cell-based therapies for the repair of damaged cartilage. Current MSC chondrogenic differentiation strategies employ large pellets; however, we have developed a technique to form small MSC aggregates (500-1,000 cells) that can reduce transport barriers while maintaining a multicellular structure analogous to cartilaginous condensations. The objective of this study was to examine the effects of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within small MSC spheroids cultured in the presence of transforming growth factor (TGF)-β1 on chondrogenesis. Spheroids with MPs induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids without MPs, although no large differences in immunostaining for these matrix molecules were observed by day 21 between these groups. Collagen I and X were also detected in the extracellular matrix (ECM) of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. This study demonstrates the use of CSMA materials to further examine the effects of matrix molecules on MSC phenotype as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific musculoskeletal tissues. © 2014 S. Karger AG, Basel.

Aerogels made of chitosan and chondroitin sulfate at high degree of neutralization: Biological properties toward wound healing.

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Concha, Miguel; Vidal, Alejandra; Giacaman, Annesi; Ojeda, Javier; Pavicic, Francisca; Oyarzun-Ampuero, Felipe A; Torres, César; Cabrera, Marcela; Moreno-Villoslada, Ignacio; Orellana, Sandra L

2018-02-09

In this study, highly neutralized, highly porous, and ultralight polymeric aerogels prepared from aqueous colloidal suspensions of chitosan (CS) and chondroitin sulfate (ChS) nanocomplexes, formulated as quasi-equimolar amounts of both, are described. These aerogels were designed as healing agents under the inspiration of minimizing the amount of matter applied to wounds, reducing the electrostatic potential of the material and avoiding covalent cross-linkers in order to decrease metabolic stress over wounds. Aerogels synthesized under these criteria are biocompatible and provide specific properties for the induction of wound healing. They do not affect neither the metabolic activity of cultured 3T3 fibroblasts nor the biochemical parameters of experimental animals, open wounds close significantly faster and, unlike control wounds, complete reepithelialization and scarring can be attained 14 days after surgery. Because of its hydration abilities, rapid adaptation to the wound bed and the early accelerator effect of wound closure, the CS/ChS aerogels appear to be functional inducers of the healing. Previous information show that CS/ChS aerogels improve wound bed quality, increase granulation tissue and have pain suppressive effect. CS/ChS aerogels are useful as safe, inexpensive and easy to handle materials for topical applications, such as skin chronic wounds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc.

Enzyme mediated synthesis of polypyrrole in the presence of chondroitin sulfate and redox mediators of natural origin

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Grijalva-Bustamante, G.A.; Evans-Villegas, A.G.; Castillo-Castro, T. del; Castillo-Ortega, M.M.; Cruz-Silva, R.; Huerta, F.; Morallón, E.

2016-01-01

Polypyrrole (PPy) was synthesized by enzyme mediated oxidation of pyrrole using naturally occurring compounds as redox mediators. The catalytic mechanism is an enzymatic cascade reaction in which hydrogen peroxide is the oxidizer and soybean peroxidase, in the presence of acetosyringone, syringaldehyde or vanillin, acts as a natural catalysts. The effect of the initial reaction composition on the polymerization yield and electrical conductivity of PPy was analyzed. Morphology of the PPy particles was studied by scanning electron microscopy and transmission electron microscopy whereas the chemical structure was studied by X-ray photoelectron and Fourier transformed infrared spectroscopic techniques. The redox mediators increased the polymerization yield without a significant modification of the electronic structure of PPy. The highest conductivity of PPy was reached when chondroitin sulfate was used simultaneously as dopant and template during pyrrole polymerization. Electroactive properties of PPy obtained from natural precursors were successfully used in the amperometric quantification of uric acid concentrations. PPy increases the amperometric sensitivity of carbon nanotube screen-printed electrodes toward uric acid detection. - Highlights: • A new method of pyrrole polymerization using naturally occurring redox mediators and doping agents was studied. • The catalytic efficiency of different redox mediators toward pyrrole oxidation was evaluated. • Two different naturally occurring polymers were studied as bifunctional steric stabilizer/doping agents. • Polypyrrole improves the amperometric response of carbon nanotube screen printed electrodes toward uric acid sensing.