chondroitinase Search Results


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  • 99
    Millipore chondroitinase abc
    Rescue of chondroitin sulfate neuroinhibition by enzymatic digestion. Representative inverted‐fluorescence confocal z‐stack projections stained for neurofilament H (NF‐H) show the growth of neurites from the original dorsal root ganglion (DRG) cell body cluster. DRGs are embedded in a hydrogel containing 2.5 mg/ml methacrylated hyaluronic acid (MAHA) adjacent to a <t>chondroitinase</t> <t>ABC</t> digested hydrogel containing 2.5 mg/ml MAHA (A), an undigested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml methacrylated chondroitin‐4‐sulfate (MACS‐A) (B), or a chondroitinase ABC digested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml MACS‐A (C). Scale bars are 0.5 mm. Neurite lengths and radial distance are measured from the edge of the inner core gel (white line(s); determined by transmitted light microscopy). Quantification of traced neurite lengths (D), radial distance of neurite extension into the inner core gel (E), and the number of neurites/groups of neurites crossing from the outer gel to the inner gel (F) demonstrate that chondroitinase ABC digestion decreases the neuroinhibition of MACS‐A because all three neurite growth measurements are higher in the digested MAHA/MACS‐A inner gels than in the undigested MAHA/MACS‐A inner gels (* p
    Chondroitinase Abc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Seikagaku chondroitinase abc
    Immature astrocytes injected with <t>chondroitinase</t> <t>ABC</t> promote regeneration of p75+ axons. Immature astrocytes were infected with a GFP lentivirus prior to transplantation into the cingulum microlesion and were therefore identified by GFP immunolabeling
    Chondroitinase Abc, supplied by Seikagaku, used in various techniques. Bioz Stars score: 92/100, based on 1391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore chondroitinase
    Immature astrocytes injected with <t>chondroitinase</t> <t>ABC</t> promote regeneration of p75+ axons. Immature astrocytes were infected with a GFP lentivirus prior to transplantation into the cingulum microlesion and were therefore identified by GFP immunolabeling
    Chondroitinase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Seikagaku chondroitinase acii
    The action of <t>chondroitinase</t> <t>ACII</t> on GAGs. CS and HA are substrates for chondroitinase ACII which acts to abstract the acidic C-5 proton (shown in red) of GlcA α to the carboxyl group. This results in cleavage of the adjacent glycosidic linkage and the formation of a ΔUA containing product and a second disaccharide product with a reducing end GalNAc residue (or GlcNAc residue in the case of HA) attached to an uronic acid residue. Since chondroitinase ACII is an exolytic enzyme it continues to cut one disaccharide at a time from the non-reducing terminus of the GAG chain. DS, containing an IdoA residue, incorrectly positions its acidic C-5 proton (shown in red) and, hence is not a substrate for chondroitinase ACII. In CS-A the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H. In CS-C the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H. In CS-D the R at positions 2 and 6 are commonly SO 3 − and the R at positions 4 is H. In CS-E the R at position 4 and 6 are commonly SO 3 − and the R at positions 2 is H. In DS the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H.
    Chondroitinase Acii, supplied by Seikagaku, used in various techniques. Bioz Stars score: 89/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    AMS Biotechnology chondroitinase abc
    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of <t>chondroitinase</t> <t>ABC-,</t> but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P
    Chondroitinase Abc, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Seikagaku protease free chondroitinase abc
    Frame A: Zymograms showing <t>chondroitinase</t> <t>ABC</t> and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa
    Protease Free Chondroitinase Abc, supplied by Seikagaku, used in various techniques. Bioz Stars score: 89/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore chondroitinase ac
    The V. fischeri aphrodisiac is a <t>chondroitinase</t> (A) Alignment of the V. fischeri EroS amino acid sequence to diverse bacterial GAG lyases reveals that V. fischeri ). Amino acids with > 50% conservation between sequences are shaded (black shading for identical amino acids and grey shading for similar amino acids. (B) Purified EroS degrades chondroitin sulfate and hyaluronan. EroS was incubated with purified chondroitin sulfate (open circle), hyaluronan (grey hexagon), dermatan sulfate (open square), and heparan sulfate (grey triangle), and GAG lyase activity of EroS was measured by monitoring the abundance of unsaturated oligosaccharide reaction products with an absorbance at 232nm. Chondroitin sulfate and hyaluronan oligosaccharides accumulated rapidly in the presence of EroS, indicating depolymerization, whereas heparan sulfate and dermatan sulfate were not depolymerized by EroS. (C) Alanine substitutions at two predicted catalytic residues in EroS (H278 and Y287) eliminated the protein’s ability to degrade chondroitin sulfate. The chondroitinase activity of either wild type EroS (open circle) or EroS-H278A,Y287A (filled circle) against purified chondroitin sulfate was measured by monitoring the abundance of unsaturated oligosaccharide products with an absorbance at 232nm. (D) The chondroitinase activity of EroS is necessary and sufficient for its function as an aphrodisiac. EroS-H278A,Y287A failed to induce swarming in S. rosetta. P. vulgaris ABC chondroitinase and F. heparinum AC chondroitinase were sufficient to induce swarming at levels similar to EroS, whereas S. hyalurolyticus hyaluronidase failed to induce swarming, indicating that chondroitinase activity is necessary and sufficient for aphrodisiac activity.
    Chondroitinase Ac, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore chondroitinase b
    The V. fischeri aphrodisiac is a <t>chondroitinase</t> (A) Alignment of the V. fischeri EroS amino acid sequence to diverse bacterial GAG lyases reveals that V. fischeri ). Amino acids with > 50% conservation between sequences are shaded (black shading for identical amino acids and grey shading for similar amino acids. (B) Purified EroS degrades chondroitin sulfate and hyaluronan. EroS was incubated with purified chondroitin sulfate (open circle), hyaluronan (grey hexagon), dermatan sulfate (open square), and heparan sulfate (grey triangle), and GAG lyase activity of EroS was measured by monitoring the abundance of unsaturated oligosaccharide reaction products with an absorbance at 232nm. Chondroitin sulfate and hyaluronan oligosaccharides accumulated rapidly in the presence of EroS, indicating depolymerization, whereas heparan sulfate and dermatan sulfate were not depolymerized by EroS. (C) Alanine substitutions at two predicted catalytic residues in EroS (H278 and Y287) eliminated the protein’s ability to degrade chondroitin sulfate. The chondroitinase activity of either wild type EroS (open circle) or EroS-H278A,Y287A (filled circle) against purified chondroitin sulfate was measured by monitoring the abundance of unsaturated oligosaccharide products with an absorbance at 232nm. (D) The chondroitinase activity of EroS is necessary and sufficient for its function as an aphrodisiac. EroS-H278A,Y287A failed to induce swarming in S. rosetta. P. vulgaris ABC chondroitinase and F. heparinum AC chondroitinase were sufficient to induce swarming at levels similar to EroS, whereas S. hyalurolyticus hyaluronidase failed to induce swarming, indicating that chondroitinase activity is necessary and sufficient for aphrodisiac activity.
    Chondroitinase B, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Seikagaku chondroitinase b
    DKO mice are DS-free. Two-day-old pups were metabolically labeled with 35 S sulfate. Skin decorin CS/DS was extracted and purified (A-C). A) CS/DS chains were analyzed on a size-permeation Superose 6 column. The HS degraded products, which were proportionally increased in the DKO mice, derive from the HS-PGs which co-eluted with decorin in the size permeation column used for PGs separation (see “ Material and methods ”). B) Split products obtained after <t>chondroitinase</t> B treatment were size separated on a Superdex Peptide column and C) the iduronic acid content was calculated. The iduronic acid analysis was conducted in duplicate samples. D) Skin PGs were extracted from E19.5 embryos of different genotypes. Decorin was stained before and after chondroitinase ABC and B treatment.
    Chondroitinase B, supplied by Seikagaku, used in various techniques. Bioz Stars score: 89/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Seikagaku chondroitinase ac
    The effect of glycosylated and non-glycosylated biglycan on DRG neurite growth. Graphs showing the mean ± S.D. of number of neurite crossings per DRG when cultured on (A ) ‘biglycan’ at a range of concentrations (2–500 µg/ml) without any enzymatic treatment; (B) ‘biglycan’ at 500 µg/ml following either <t>chondroitinase</t> ABC or chondroitinase AC digestion (both at 250 mU/ml) or in buffer; * P
    Chondroitinase Ac, supplied by Seikagaku, used in various techniques. Bioz Stars score: 91/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Seikagaku chondroitinase
    ( A ) NRP1 is GAG modified on a single Ser 612 residue. CASMCs were transfected with adenoviral vectors encoding WT or S612A mutant NRP1. NRP1 S612A is not GAG modified. ( B ) Multiple alignments of NRP1 from different species. Ser 612 is highly conserved among vertebrates. ( C ) NRP2, an NRP family member, is not GAG modified. ( D ) Design of siRNA and adenovirus constructs. Ser 612 exists in the bridge region between the b1b2 and MAM domains. ( E ) Replacement of Ser 612 by Ala 612 of NRP1 did not change binding to VEGF. Cos7 cells were transfected with either NRP1 WT′ or S612A expression vector and preincubated with heparitinase (1.5 mU/ml), heparinase (1.5 mU/ml), and <t>chondroitinase</t> (20 mU/ml) in the culture medium at 37°C for 2 h to make NRP1 non-GAG form. After incubation with 125 I-labeled VEGF for 30 min at room temperature, cell lysates were immunoprecipitated by anti-NRP1 antibody, and the bound radioactivity was quantitated using a gamma counter. Data are from three independent experiments. For panel E, error bars represent s.e.
    Chondroitinase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 90/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    IBEX Technologies chondroitinase b
    Pharmacological knockdown of other glycosaminoglycan (GAG) family members does not affect Aβ 1-40 -mediated ROS production in VSMC. Primary human cerebral VSMC were pre-treated with <t>chondroitinase</t> B (10 -1 IU/mL; selectively degrades dermatin sulfate; panel a ), chondroitinase AC (10 -1 IU/mL; selectively degrades chondroitin sulfate; panel a ), or varying concentrations of the sulfation inhibitor sodium chlorate (5-50 mM; panel b ) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM), and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. Results are representative of 3 independent experiments performed in triplicate. *p
    Chondroitinase B, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 89/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim chondroitinase abc
    Pharmacological knockdown of other glycosaminoglycan (GAG) family members does not affect Aβ 1-40 -mediated ROS production in VSMC. Primary human cerebral VSMC were pre-treated with <t>chondroitinase</t> B (10 -1 IU/mL; selectively degrades dermatin sulfate; panel a ), chondroitinase AC (10 -1 IU/mL; selectively degrades chondroitin sulfate; panel a ), or varying concentrations of the sulfation inhibitor sodium chlorate (5-50 mM; panel b ) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM), and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. Results are representative of 3 independent experiments performed in triplicate. *p
    Chondroitinase Abc, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    AMS Biotechnology chondroitinase enzyme
    VP16-KLF7 and <t>Lenti-chondroitinase</t> increase the proximity of injured sensory axons to sites of spinal injury but do not promote regeneration beyond the injury Sensory neurons in adult mice were transduced by lumbar puncture delivery of AAV-EBFP-2A-mCherry or AAV-VP16-2A-mCherry by lumbar puncture, and received cervical dorsal hemisections accompanied by either control injections or spinal injections of Lenti-Chase. Four weeks later ascending sensory axons were labeled by injection of Dextran-Alexafluor 488 injections to the sciatic nerve, and then sacrificed two weeks later. ( A–D) show sagittal sections of spinal cord with GFAP (blue) outlining the site of spinal transection (arrowhead). Ascending Dextran-labeled sensory axons (arrow) are green. Compared to controls (A), individual treatment with VP16-KLF7 (B) and Lenti-Chase (C) resulted in axon growth that approached nearer to the lesion center. Combined VP16KLF7 and Lenti Chase (D) appeared similar to the individual treatments. E. Quantification of the minimum distance between the lesion center and the nearest axon showed a significant reduction from control in both VP16KLF7 and Lenti-Chase treatments, without significant additive effects. N≥6 animals per group, **p
    Chondroitinase Enzyme, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant chondroitinase abc
    Effects of increased NG2 expression on neurite growth. A–D , Comparison of the level of NG2 expression in control NG2 cells and in NG2 cells infected with adeno-NG2. A , B , Surface immunofluorescence labeling (live-cell labeling) for NG2 on uninfected ( A ) and adeno-NG2-infected cells ( B ; moi 5). Images were acquired under identical exposure conditions below pixel saturation. Scale bar: (in B ) A , B , 12 μm. C , Quantification of fluorescence intensity of uninfected NG2 cells and NG2 cells infected with adeno-NG2. Cells transduced with adeno-NG2 expressed fivefold more surface NG2 than uninfected NG2 cells. D , Western blots comparing the amount of NG2 in extracts of uninfected and adeno-NG2-infected NG2 cells. The same blot was probed with antibodies to NG2 (top) and GAPDH (bottom) chABC+: extracts treated with <t>chondroitinase</t> <t>ABC.</t> Smaller amounts of protein extracts from adeno-NG2-infected cells were loaded to allow for densitometric analyses of the samples within the dynamic ranges of the pixels, as seen by the weaker GAPDH bands in lanes 3 and 4. The positions of molecular weight standards are indicated on the left. E–I , Response of P1 hippocampal neurons to NG2 cells expressing elevated levels of NG2. E , F , Double-immunofluorescence labeling for NG2 (red) and βIII tubulin (green) to illustrate the spatial relationship between neurites and NG2 cells expressing normal ( E ) or increased levels of surface NG2 after adeno-NG2 transduction ( F ; moi 5). Scale bar: (in F ) E , F , 24 μm. G , Percentage of neurite tips contacting uninfected or adeno-NG2-infected NG2 cells. ns, No statistically significant difference between the number of neurite tips contacting uninfected and adeno-NG2-infected NG2 cells ( P = 0.239, t test). H , Total neurite lengths of neurons grown on uninfected or adeno-NG2-infected NG2 cells. ns, No significant difference ( P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL ( P
    Chondroitinase Abc, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Rescue of chondroitin sulfate neuroinhibition by enzymatic digestion. Representative inverted‐fluorescence confocal z‐stack projections stained for neurofilament H (NF‐H) show the growth of neurites from the original dorsal root ganglion (DRG) cell body cluster. DRGs are embedded in a hydrogel containing 2.5 mg/ml methacrylated hyaluronic acid (MAHA) adjacent to a chondroitinase ABC digested hydrogel containing 2.5 mg/ml MAHA (A), an undigested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml methacrylated chondroitin‐4‐sulfate (MACS‐A) (B), or a chondroitinase ABC digested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml MACS‐A (C). Scale bars are 0.5 mm. Neurite lengths and radial distance are measured from the edge of the inner core gel (white line(s); determined by transmitted light microscopy). Quantification of traced neurite lengths (D), radial distance of neurite extension into the inner core gel (E), and the number of neurites/groups of neurites crossing from the outer gel to the inner gel (F) demonstrate that chondroitinase ABC digestion decreases the neuroinhibition of MACS‐A because all three neurite growth measurements are higher in the digested MAHA/MACS‐A inner gels than in the undigested MAHA/MACS‐A inner gels (* p

    Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

    Article Title: Development of an In Vitro Intervertebral Disc Innervation Model to Screen Neuroinhibitory Biomaterials

    doi: 10.1002/jor.24557

    Figure Lengend Snippet: Rescue of chondroitin sulfate neuroinhibition by enzymatic digestion. Representative inverted‐fluorescence confocal z‐stack projections stained for neurofilament H (NF‐H) show the growth of neurites from the original dorsal root ganglion (DRG) cell body cluster. DRGs are embedded in a hydrogel containing 2.5 mg/ml methacrylated hyaluronic acid (MAHA) adjacent to a chondroitinase ABC digested hydrogel containing 2.5 mg/ml MAHA (A), an undigested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml methacrylated chondroitin‐4‐sulfate (MACS‐A) (B), or a chondroitinase ABC digested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml MACS‐A (C). Scale bars are 0.5 mm. Neurite lengths and radial distance are measured from the edge of the inner core gel (white line(s); determined by transmitted light microscopy). Quantification of traced neurite lengths (D), radial distance of neurite extension into the inner core gel (E), and the number of neurites/groups of neurites crossing from the outer gel to the inner gel (F) demonstrate that chondroitinase ABC digestion decreases the neuroinhibition of MACS‐A because all three neurite growth measurements are higher in the digested MAHA/MACS‐A inner gels than in the undigested MAHA/MACS‐A inner gels (* p

    Article Snippet: Gels were washed with HBSS (BW10–543F; Thermo Fisher Scientific), incubated with 2.5U chondroitinase ABC (C3667; MilliporeSigma) in 2 ml HBSS plus 61 mM sodium acetate (W302406; MilliporeSigma) for 3 h at 37°C, and washed 9× 15 min with PBS before being stored in PBS at 4°C overnight.

    Techniques: Fluorescence, Staining, Magnetic Cell Separation, Light Microscopy

    Enzymatic treatment with chondroitinase ABC eliminates Wisteria floribunda agglutinin (WFA) labeling in perineuronal nets (PNNs) and glial cells. Photomicrographs show neuronal clusters in layer II of the entorhinal cortex in sections processed for WFA

    Journal: Archives of general psychiatry

    Article Title: Extracellular Matrix-Glial Abnormalities in the Amygdala and Entorhinal Cortex of Subjects Diagnosed With Schizophrenia

    doi: 10.1001/archgenpsychiatry.2009.196

    Figure Lengend Snippet: Enzymatic treatment with chondroitinase ABC eliminates Wisteria floribunda agglutinin (WFA) labeling in perineuronal nets (PNNs) and glial cells. Photomicrographs show neuronal clusters in layer II of the entorhinal cortex in sections processed for WFA

    Article Snippet: Following antigen unmasking with citric acid buffer, as above, sections were incubated overnight at 37°C in chondroitinase buffer (1:8 chondroitinase ABC, 10 mU/μL, C-2905 [Sigma-Aldrich] in 50mM Tris, pH 8.0, 60mM sodium acetate, 0.02% bovine albumin serum).

    Techniques: Labeling

    Immature astrocytes injected with chondroitinase ABC promote regeneration of p75+ axons. Immature astrocytes were infected with a GFP lentivirus prior to transplantation into the cingulum microlesion and were therefore identified by GFP immunolabeling

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: Immature astrocytes injected with chondroitinase ABC promote regeneration of p75+ axons. Immature astrocytes were infected with a GFP lentivirus prior to transplantation into the cingulum microlesion and were therefore identified by GFP immunolabeling

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques: Injection, Infection, Transplantation Assay, Immunolabeling

    Quantification of axonal regeneration demonstrates that immature astrocytes combined with chondroitinase ABC would be the best treatment for brain injuries. Axonal regeneration was quantified by counting the number of axons crossing the proximal edge

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: Quantification of axonal regeneration demonstrates that immature astrocytes combined with chondroitinase ABC would be the best treatment for brain injuries. Axonal regeneration was quantified by counting the number of axons crossing the proximal edge

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques:

    Pre-treatment of gradient spots with chondroitinase ABC enhances axon rim crossing of DRG neurons cultured on immature astrocytes. Adult DRG neurons were cultured for 2 days on immature (A–C) or mature astrocytes (D–F) which had been plated

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: Pre-treatment of gradient spots with chondroitinase ABC enhances axon rim crossing of DRG neurons cultured on immature astrocytes. Adult DRG neurons were cultured for 2 days on immature (A–C) or mature astrocytes (D–F) which had been plated

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques: Cell Culture

    The p75+ axons in chondroitinase ABC treated microlesions are not capable of regenerating. Microlesions were performed in the cingulum and treated with a single injection of 5U/ml chondroitinase ABC. Chondroitinase treatment resulted in 2B6 immunolabelling

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: The p75+ axons in chondroitinase ABC treated microlesions are not capable of regenerating. Microlesions were performed in the cingulum and treated with a single injection of 5U/ml chondroitinase ABC. Chondroitinase treatment resulted in 2B6 immunolabelling

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques: Injection

    Immature astrocytes are capable of growing on high levels of CSPGs. Immature and mature astrocytes were cultured on gradient spots of a mixture of CSPG and laminin for 1 day, 3 days and 5 days (A), or on spots pre-treated with chondroitinase ABC for 5

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: Immature astrocytes are capable of growing on high levels of CSPGs. Immature and mature astrocytes were cultured on gradient spots of a mixture of CSPG and laminin for 1 day, 3 days and 5 days (A), or on spots pre-treated with chondroitinase ABC for 5

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques: Cell Culture

    The action of chondroitinase ACII on GAGs. CS and HA are substrates for chondroitinase ACII which acts to abstract the acidic C-5 proton (shown in red) of GlcA α to the carboxyl group. This results in cleavage of the adjacent glycosidic linkage and the formation of a ΔUA containing product and a second disaccharide product with a reducing end GalNAc residue (or GlcNAc residue in the case of HA) attached to an uronic acid residue. Since chondroitinase ACII is an exolytic enzyme it continues to cut one disaccharide at a time from the non-reducing terminus of the GAG chain. DS, containing an IdoA residue, incorrectly positions its acidic C-5 proton (shown in red) and, hence is not a substrate for chondroitinase ACII. In CS-A the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H. In CS-C the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H. In CS-D the R at positions 2 and 6 are commonly SO 3 − and the R at positions 4 is H. In CS-E the R at position 4 and 6 are commonly SO 3 − and the R at positions 2 is H. In DS the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H.

    Journal: Biotechnology journal

    Article Title: Cloning and expression of recombinant chondroitinase AC II and its comparison to the Arthrobacter aurescens enzyme

    doi: 10.1002/biot.201700239

    Figure Lengend Snippet: The action of chondroitinase ACII on GAGs. CS and HA are substrates for chondroitinase ACII which acts to abstract the acidic C-5 proton (shown in red) of GlcA α to the carboxyl group. This results in cleavage of the adjacent glycosidic linkage and the formation of a ΔUA containing product and a second disaccharide product with a reducing end GalNAc residue (or GlcNAc residue in the case of HA) attached to an uronic acid residue. Since chondroitinase ACII is an exolytic enzyme it continues to cut one disaccharide at a time from the non-reducing terminus of the GAG chain. DS, containing an IdoA residue, incorrectly positions its acidic C-5 proton (shown in red) and, hence is not a substrate for chondroitinase ACII. In CS-A the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H. In CS-C the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H. In CS-D the R at positions 2 and 6 are commonly SO 3 − and the R at positions 4 is H. In CS-E the R at position 4 and 6 are commonly SO 3 − and the R at positions 2 is H. In DS the R at position 4 is commonly SO 3 − and the R at positions 2 and 6 is H.

    Article Snippet: KS-branched CS has been found to be resistant to degradation by both chondroitinase ABC and chondroitinase ACII at very low concentrations, but when treated with Seikagaku’s chondroitinase ACII at a concentration of 12.5 mU, several unidentified peaks were observed on the reaction’s anion-exchange chromatogram [ ].

    Techniques:

    The SDS-PAGE analysis of chondroitinase ACII enzymes with the ladder labeled L for each gel. Panel A gel result shows the induced and uninduced fractions of the tA16ACII and tA16ACII(I236T) enzymes expressed in E. coli BL21. The theoretical molecular weight was predicted to be 83.5 kDa and in the gel, the approximate molecular weight is ~75 kDa. The lanes are - induced tA16ACII(I236T): 1a, uninduced tA16ACII(I236T): 2a, induced tA16ACII: 3a, uninduced tA16ACII: 4a. Panel B gel result shows the tA16ACII and tA16ACII(I236T) enzymes expressed in E. coli BL21 and purified from Nickel column, with the tA16ACII(I236T) enzyme in the lanes on the left side of the gel and tA16ACII on the right side. The lanes are – 1b 5b: soluble fraction, 2b 6b: insoluble fraction, 3b 7b: flow through, 4b 8b: first wash. Panel C shows the gel result of the purified recombinant enzymes after buffer exchange in a 10 kDa spin column, with the lanes labeled – 1c: tA16ACII, 2c: tA16ACII(I236T). Panel D gel result shows the A16ACII enzyme (which has a theoretical molecular weight of ~76 kDa), among other proteins in the crude supernatant. The lanes are labeled – 1d: A16ACII grown in LB supplemented with 0.2% CS-A: A and 2d: A16ACII grown in LB supplemented with 0.2% CS-C.

    Journal: Biotechnology journal

    Article Title: Cloning and expression of recombinant chondroitinase AC II and its comparison to the Arthrobacter aurescens enzyme

    doi: 10.1002/biot.201700239

    Figure Lengend Snippet: The SDS-PAGE analysis of chondroitinase ACII enzymes with the ladder labeled L for each gel. Panel A gel result shows the induced and uninduced fractions of the tA16ACII and tA16ACII(I236T) enzymes expressed in E. coli BL21. The theoretical molecular weight was predicted to be 83.5 kDa and in the gel, the approximate molecular weight is ~75 kDa. The lanes are - induced tA16ACII(I236T): 1a, uninduced tA16ACII(I236T): 2a, induced tA16ACII: 3a, uninduced tA16ACII: 4a. Panel B gel result shows the tA16ACII and tA16ACII(I236T) enzymes expressed in E. coli BL21 and purified from Nickel column, with the tA16ACII(I236T) enzyme in the lanes on the left side of the gel and tA16ACII on the right side. The lanes are – 1b 5b: soluble fraction, 2b 6b: insoluble fraction, 3b 7b: flow through, 4b 8b: first wash. Panel C shows the gel result of the purified recombinant enzymes after buffer exchange in a 10 kDa spin column, with the lanes labeled – 1c: tA16ACII, 2c: tA16ACII(I236T). Panel D gel result shows the A16ACII enzyme (which has a theoretical molecular weight of ~76 kDa), among other proteins in the crude supernatant. The lanes are labeled – 1d: A16ACII grown in LB supplemented with 0.2% CS-A: A and 2d: A16ACII grown in LB supplemented with 0.2% CS-C.

    Article Snippet: KS-branched CS has been found to be resistant to degradation by both chondroitinase ABC and chondroitinase ACII at very low concentrations, but when treated with Seikagaku’s chondroitinase ACII at a concentration of 12.5 mU, several unidentified peaks were observed on the reaction’s anion-exchange chromatogram [ ].

    Techniques: SDS Page, Labeling, Molecular Weight, Purification, Nickel Column, Flow Cytometry, Recombinant, Buffer Exchange

    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Journal: Cell Research

    Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

    doi: 10.1038/cr.2015.29

    Figure Lengend Snippet: Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Article Snippet: Western blot To allow detection of full-length syndecan core proteins, the samples were digested with (0.006 U/ml) heparitinase (Amsbio) and (0.166 U/ml) chondroitinase ABC (Amsbio) for 3 h at 37 °C in heparitinase buffer (0.1% Triton, 0.1 M NaCl, 1 mM CaCl2, 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7).

    Techniques: Activity Assay, Migration, Molecular Weight, Western Blot, Derivative Assay

    Heparanase acts through syntenin-1, ALIX and the syntenin-ALIX interaction to stimulate intraluminal budding. (A) The role of syntenin-1 and (B) the role of ALIX in the effect of heparanase on exosomes were investigated by the knockdown of syntenin-1 and ALIX (using Synt1 RNAi and ALIX RNAi, respectively) in MCF-7 cells. Non-targeting RNAi (NT) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both the experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of two independent experiments. Note that lysate and exosome samples derived from NT and KD cells, separated by a blank space in A , were run in the same gel, but not side by side, and that the band intensities in each row are directly comparable. (C) Confocal micrographs of MCF-7 cells co-transfected with wild-type mCherry-syntenin-1 (mCherry-Synt1 WT) or with mCherry-syntenin-1 harboring mutant LYP sequences and therefore defective in ALIX-binding (mCherry-Synt1-ΔALIX; red in merge), Cerulean-RAB5 Q79L (blue in merge) and syndecan-1 (green in merge). Intraluminal budding of wild-type and mutant mCherry-syntenin-1 and of syndecan-1 cytoplasmic domain was scored in the presence (Hep) or absence (no Hep) of heparanase. (D) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge) and Cerulean-RAB5 Q79L (blue in merge). In addition, the cells were transfected with RNAi targeting ALIX ( ALIX RNAi) or with non-targeting RNAi (NT RNAi). Intraluminal budding of mCherry-syntenin-1 was evaluated in the presence (Hep) or absence (no Hep) of heparanase. (E) Quantification of intraluminal budding of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain, as in C and (F) quantitation of intraluminal budding of mCherry-syntenin-1, as in D , measuring the mean fluorescence intensity of mCherry-syntenin-1 and syndecan-1 in the lumen of RAB5 Q79L -positive endosomes (mean gray value per pixel). Bar heights represent mean values calculated from three independent experiments, scoring at least 30 cells per experiment. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Journal: Cell Research

    Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

    doi: 10.1038/cr.2015.29

    Figure Lengend Snippet: Heparanase acts through syntenin-1, ALIX and the syntenin-ALIX interaction to stimulate intraluminal budding. (A) The role of syntenin-1 and (B) the role of ALIX in the effect of heparanase on exosomes were investigated by the knockdown of syntenin-1 and ALIX (using Synt1 RNAi and ALIX RNAi, respectively) in MCF-7 cells. Non-targeting RNAi (NT) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both the experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of two independent experiments. Note that lysate and exosome samples derived from NT and KD cells, separated by a blank space in A , were run in the same gel, but not side by side, and that the band intensities in each row are directly comparable. (C) Confocal micrographs of MCF-7 cells co-transfected with wild-type mCherry-syntenin-1 (mCherry-Synt1 WT) or with mCherry-syntenin-1 harboring mutant LYP sequences and therefore defective in ALIX-binding (mCherry-Synt1-ΔALIX; red in merge), Cerulean-RAB5 Q79L (blue in merge) and syndecan-1 (green in merge). Intraluminal budding of wild-type and mutant mCherry-syntenin-1 and of syndecan-1 cytoplasmic domain was scored in the presence (Hep) or absence (no Hep) of heparanase. (D) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge) and Cerulean-RAB5 Q79L (blue in merge). In addition, the cells were transfected with RNAi targeting ALIX ( ALIX RNAi) or with non-targeting RNAi (NT RNAi). Intraluminal budding of mCherry-syntenin-1 was evaluated in the presence (Hep) or absence (no Hep) of heparanase. (E) Quantification of intraluminal budding of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain, as in C and (F) quantitation of intraluminal budding of mCherry-syntenin-1, as in D , measuring the mean fluorescence intensity of mCherry-syntenin-1 and syndecan-1 in the lumen of RAB5 Q79L -positive endosomes (mean gray value per pixel). Bar heights represent mean values calculated from three independent experiments, scoring at least 30 cells per experiment. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Article Snippet: Western blot To allow detection of full-length syndecan core proteins, the samples were digested with (0.006 U/ml) heparitinase (Amsbio) and (0.166 U/ml) chondroitinase ABC (Amsbio) for 3 h at 37 °C in heparitinase buffer (0.1% Triton, 0.1 M NaCl, 1 mM CaCl2, 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7).

    Techniques: Activity Assay, Migration, Molecular Weight, Western Blot, Derivative Assay, Transfection, Mutagenesis, Binding Assay, Quantitation Assay, Fluorescence

    Heparanase stimulates the production of syntenin-1-containing exosomes. (A) Exosome production was evaluated after overnight conditioning of MCF-7 cells with increasing concentrations of proheparanase (0.04-25 nM) and compared to that of cells not receiving proheparanase (0 nM). Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. For each condition both the lysate and exosomal fractions were analyzed by western blot, using cognate antibodies against heparanase, monitoring the conversion of proheparanase (Prohep) into mature heparanase (Hep) and against different exosomal markers: syntenin-1 (Synt1), syndecan-1 (SDC1), syndecan-4 (SDC4), CD63, flotillin-1 (Flo1), CD9 and CD81. Syndecan-1, which is a hybrid heparan sulfate (HS)/chondroitin sulfate proteoglycan, was analyzed using two different approaches. In one approach, the samples were digested with both heparitinase and chondroitinase ABC, removing all glycosaminoglycan chains and enabling visualization of the full-length syndecan core proteins (SDC1 FL) as sharp bands. In the other approach, the samples were digested with chondroitinase ABC only, leaving the HS on the syndecans (SDC1 with HS); comparison of 'SDC1 with HS' and 'SDC1 FL' yields information on the mass of HS on syndecans. Because of the heterogeneity in HS chain length, syndecan-1 with HS is smeared over a wide mass range in the absence of heparanase activity (and is therefore hardly visible in western blot, as illustrated by lane 1 of the lysates). With increasing heparanase activity, the HS chains on syndecan-1 are trimmed to shorter chains of more or less the same length, syndecan-1 with HS migrating as one or a few bands that are readily visualized in western blot (as illustrated by lane 6 of the lysates). Note that cell lysates contain mainly full-length syndecan core proteins; the opposite is true for exosomes, where hardly any full-length syndecan is detected and C-terminal fragments (CTFs) represent the dominant form. β-actin was used as a loading control for the lysates. Western blots are representative of five independent experiments. (B) Histogram representing the quantification of the exosomal levels of syntenin-1 (Synt1), syndecan-1 CTF (SDC1 CTF), CD63, syndecan-4 CTF (SDC4 CTF) and flotillin-1 (Flo1) in response to the addition of increasing concentrations (0 nM till 25 nM) of proheparanase. Values are relative to the exosomal levels measured in absence of exogenously added proheparanase. Bar heights represent mean values, calculated from five independent experiments. Individual data points are shown as white dots on top of the corresponding bars. * P

    Journal: Cell Research

    Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

    doi: 10.1038/cr.2015.29

    Figure Lengend Snippet: Heparanase stimulates the production of syntenin-1-containing exosomes. (A) Exosome production was evaluated after overnight conditioning of MCF-7 cells with increasing concentrations of proheparanase (0.04-25 nM) and compared to that of cells not receiving proheparanase (0 nM). Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. For each condition both the lysate and exosomal fractions were analyzed by western blot, using cognate antibodies against heparanase, monitoring the conversion of proheparanase (Prohep) into mature heparanase (Hep) and against different exosomal markers: syntenin-1 (Synt1), syndecan-1 (SDC1), syndecan-4 (SDC4), CD63, flotillin-1 (Flo1), CD9 and CD81. Syndecan-1, which is a hybrid heparan sulfate (HS)/chondroitin sulfate proteoglycan, was analyzed using two different approaches. In one approach, the samples were digested with both heparitinase and chondroitinase ABC, removing all glycosaminoglycan chains and enabling visualization of the full-length syndecan core proteins (SDC1 FL) as sharp bands. In the other approach, the samples were digested with chondroitinase ABC only, leaving the HS on the syndecans (SDC1 with HS); comparison of 'SDC1 with HS' and 'SDC1 FL' yields information on the mass of HS on syndecans. Because of the heterogeneity in HS chain length, syndecan-1 with HS is smeared over a wide mass range in the absence of heparanase activity (and is therefore hardly visible in western blot, as illustrated by lane 1 of the lysates). With increasing heparanase activity, the HS chains on syndecan-1 are trimmed to shorter chains of more or less the same length, syndecan-1 with HS migrating as one or a few bands that are readily visualized in western blot (as illustrated by lane 6 of the lysates). Note that cell lysates contain mainly full-length syndecan core proteins; the opposite is true for exosomes, where hardly any full-length syndecan is detected and C-terminal fragments (CTFs) represent the dominant form. β-actin was used as a loading control for the lysates. Western blots are representative of five independent experiments. (B) Histogram representing the quantification of the exosomal levels of syntenin-1 (Synt1), syndecan-1 CTF (SDC1 CTF), CD63, syndecan-4 CTF (SDC4 CTF) and flotillin-1 (Flo1) in response to the addition of increasing concentrations (0 nM till 25 nM) of proheparanase. Values are relative to the exosomal levels measured in absence of exogenously added proheparanase. Bar heights represent mean values, calculated from five independent experiments. Individual data points are shown as white dots on top of the corresponding bars. * P

    Article Snippet: Western blot To allow detection of full-length syndecan core proteins, the samples were digested with (0.006 U/ml) heparitinase (Amsbio) and (0.166 U/ml) chondroitinase ABC (Amsbio) for 3 h at 37 °C in heparitinase buffer (0.1% Triton, 0.1 M NaCl, 1 mM CaCl2, 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7).

    Techniques: Western Blot, Activity Assay

    The effect of heparanase on exosome production depends on the modification of the heparan sulfate (HS) on syndecans. (A) The role of heparanase enzymatic activity on exosome production was investigated by comparing MCF-7 cells stably expressing wild-type heparanase (WT), catalytically dead heparanase (Cat) or empty vector (Φ) in western blot, in the absence (−) or presence (+) of 10 nM exogenously added proheparanase. (B) The importance of HS was analyzed by treating MCF-7 cells with RNAi targeting EXT1 and EXT2 (KD). Non-targeting RNAi (NT) was used as a control. Cells were challenged with 10 nM proheparanase (+) or left untreated (−). Heparanase activity, reducing the HS on syndecan, was apparent from the migration of chondroitinase ABC-treated syndecan-1 present in cell lysates (SDC1 with HS). EXT1 and EXT2 knockdown leads to the appearance of syndecan-1 that is not substituted with HS (a band running slightly > 70 kDa, after chondroitinase ABC digestion only), not detectable in cells treated with non-targeting RNAi, where all syndecan is substituted with HS (and is larger than SDC FL). A small amount of the syndecan-1 still carried HS and was affected by heparanase addition (yielding a band slightly > 100 kDa, after chondroitinase ABC digestion only), indicating an incomplete knockdown of EXT1 and EXT2. Western blots are representative of three independent experiments. (C) Quantification of the effect of EXT1 and EXT2 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF, CD63 and flotillin-1 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi and heparanase, dark gray bars; EXT1 and EXT2 RNAi, light gray bars; EXT1 and EXT2 RNAi and heparanase, white bars). Values are relative to the exosomal levels measured in cells treated with non-targeting RNAi and in the absence of exogenously added proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Journal: Cell Research

    Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

    doi: 10.1038/cr.2015.29

    Figure Lengend Snippet: The effect of heparanase on exosome production depends on the modification of the heparan sulfate (HS) on syndecans. (A) The role of heparanase enzymatic activity on exosome production was investigated by comparing MCF-7 cells stably expressing wild-type heparanase (WT), catalytically dead heparanase (Cat) or empty vector (Φ) in western blot, in the absence (−) or presence (+) of 10 nM exogenously added proheparanase. (B) The importance of HS was analyzed by treating MCF-7 cells with RNAi targeting EXT1 and EXT2 (KD). Non-targeting RNAi (NT) was used as a control. Cells were challenged with 10 nM proheparanase (+) or left untreated (−). Heparanase activity, reducing the HS on syndecan, was apparent from the migration of chondroitinase ABC-treated syndecan-1 present in cell lysates (SDC1 with HS). EXT1 and EXT2 knockdown leads to the appearance of syndecan-1 that is not substituted with HS (a band running slightly > 70 kDa, after chondroitinase ABC digestion only), not detectable in cells treated with non-targeting RNAi, where all syndecan is substituted with HS (and is larger than SDC FL). A small amount of the syndecan-1 still carried HS and was affected by heparanase addition (yielding a band slightly > 100 kDa, after chondroitinase ABC digestion only), indicating an incomplete knockdown of EXT1 and EXT2. Western blots are representative of three independent experiments. (C) Quantification of the effect of EXT1 and EXT2 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF, CD63 and flotillin-1 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi and heparanase, dark gray bars; EXT1 and EXT2 RNAi, light gray bars; EXT1 and EXT2 RNAi and heparanase, white bars). Values are relative to the exosomal levels measured in cells treated with non-targeting RNAi and in the absence of exogenously added proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Article Snippet: Western blot To allow detection of full-length syndecan core proteins, the samples were digested with (0.006 U/ml) heparitinase (Amsbio) and (0.166 U/ml) chondroitinase ABC (Amsbio) for 3 h at 37 °C in heparitinase buffer (0.1% Triton, 0.1 M NaCl, 1 mM CaCl2, 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7).

    Techniques: Modification, Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Migration

    Frame A: Zymograms showing chondroitinase ABC and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa

    Journal: Archives of biochemistry and biophysics

    Article Title: Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

    doi: 10.1016/j.abb.2012.02.003

    Figure Lengend Snippet: Frame A: Zymograms showing chondroitinase ABC and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa

    Article Snippet: 230 kDa enzyme was digested with protease-free chondroitinase ABC (40 μU) (Seikagaku Co., Tokyo, Japan) in chondroitinase buffer containing 50 mM Tris, pH 8.0, 30 mM sodium acetate, 0.02% (v/v) Brij-35, 0.02% (w/v) NaN3 and 0.05% (w/v) protease free BSA at 37°C for 2 h. Digested proteins were separated by SDS-PAGE and detected by Coomassie colloidal blue stain (OWL Separation Systems, Woburn, MA).

    Techniques: Mobility Shift

    MMP enzyme assay using peptide substrate. The effects of APMA activation and chondroitinase ABC treatment on the enzyme activity of the 230 kDa gelatinase were tested using a quenched fluorescent synthetic peptide substrate. APMA activated the latent

    Journal: Archives of biochemistry and biophysics

    Article Title: Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

    doi: 10.1016/j.abb.2012.02.003

    Figure Lengend Snippet: MMP enzyme assay using peptide substrate. The effects of APMA activation and chondroitinase ABC treatment on the enzyme activity of the 230 kDa gelatinase were tested using a quenched fluorescent synthetic peptide substrate. APMA activated the latent

    Article Snippet: 230 kDa enzyme was digested with protease-free chondroitinase ABC (40 μU) (Seikagaku Co., Tokyo, Japan) in chondroitinase buffer containing 50 mM Tris, pH 8.0, 30 mM sodium acetate, 0.02% (v/v) Brij-35, 0.02% (w/v) NaN3 and 0.05% (w/v) protease free BSA at 37°C for 2 h. Digested proteins were separated by SDS-PAGE and detected by Coomassie colloidal blue stain (OWL Separation Systems, Woburn, MA).

    Techniques: Enzymatic Assay, Activation Assay, Activity Assay

    Macromolecular composition of the 230 kDa gelatinase. Frame A shows a chondroitinase ABC digestion of the 230 kDa gelatinase on SDS-PAGE stained with Coomassie colloidal blue stain. Lanes M, molecular weight standards; 1, chondroitinase ABC enzyme alone;

    Journal: Archives of biochemistry and biophysics

    Article Title: Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

    doi: 10.1016/j.abb.2012.02.003

    Figure Lengend Snippet: Macromolecular composition of the 230 kDa gelatinase. Frame A shows a chondroitinase ABC digestion of the 230 kDa gelatinase on SDS-PAGE stained with Coomassie colloidal blue stain. Lanes M, molecular weight standards; 1, chondroitinase ABC enzyme alone;

    Article Snippet: 230 kDa enzyme was digested with protease-free chondroitinase ABC (40 μU) (Seikagaku Co., Tokyo, Japan) in chondroitinase buffer containing 50 mM Tris, pH 8.0, 30 mM sodium acetate, 0.02% (v/v) Brij-35, 0.02% (w/v) NaN3 and 0.05% (w/v) protease free BSA at 37°C for 2 h. Digested proteins were separated by SDS-PAGE and detected by Coomassie colloidal blue stain (OWL Separation Systems, Woburn, MA).

    Techniques: SDS Page, Staining, Molecular Weight

    Influence of TIMPs and chondroitinase ABC treatment on 230 kDa gelatinase activity. In all assays the 230 kDa gelatinase was APMA activated and incubated with the peptide substrate in presence of 500 ng of TIMP-1 or TIMP-2 either without (frame A) or

    Journal: Archives of biochemistry and biophysics

    Article Title: Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

    doi: 10.1016/j.abb.2012.02.003

    Figure Lengend Snippet: Influence of TIMPs and chondroitinase ABC treatment on 230 kDa gelatinase activity. In all assays the 230 kDa gelatinase was APMA activated and incubated with the peptide substrate in presence of 500 ng of TIMP-1 or TIMP-2 either without (frame A) or

    Article Snippet: 230 kDa enzyme was digested with protease-free chondroitinase ABC (40 μU) (Seikagaku Co., Tokyo, Japan) in chondroitinase buffer containing 50 mM Tris, pH 8.0, 30 mM sodium acetate, 0.02% (v/v) Brij-35, 0.02% (w/v) NaN3 and 0.05% (w/v) protease free BSA at 37°C for 2 h. Digested proteins were separated by SDS-PAGE and detected by Coomassie colloidal blue stain (OWL Separation Systems, Woburn, MA).

    Techniques: Activity Assay, Incubation

    GPC1 expression in tumor xenografts. ( A ) Immunoblotting. Tumors from sham-transfected and GAS PANC-1 cells (GAS6 and GAS7) were homogenized and incubated with control buffer, heparitinase, or heparitinase and chondroitinase ABC (Ch-ABC). Protein lysates were subjected to immunoblotting using affinity-purified rabbit anti-GPC1 antibody as reported in Methods. Membranes from cell lysates were reprobed with an anti-ERK2 antibody to confirm equivalent loading of lanes. ( B ) Densitometric analysis. Frozen lysates for immunoblotting from 6 sham, 5 GAS6, and 4 GAS7 tumors were subjected to densitometry. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells

    doi: 10.1172/JCI32412

    Figure Lengend Snippet: GPC1 expression in tumor xenografts. ( A ) Immunoblotting. Tumors from sham-transfected and GAS PANC-1 cells (GAS6 and GAS7) were homogenized and incubated with control buffer, heparitinase, or heparitinase and chondroitinase ABC (Ch-ABC). Protein lysates were subjected to immunoblotting using affinity-purified rabbit anti-GPC1 antibody as reported in Methods. Membranes from cell lysates were reprobed with an anti-ERK2 antibody to confirm equivalent loading of lanes. ( B ) Densitometric analysis. Frozen lysates for immunoblotting from 6 sham, 5 GAS6, and 4 GAS7 tumors were subjected to densitometry. * P

    Article Snippet: The following materials were purchased from the following companies: DMEM, RPMI 1640, and trypsin-EDTA from Mediatech Inc.; FBS from Omega Scientific Inc.; G418 from GIBCO Laboratories; Noble agar from Difco Laboratories; recombinant human EGF from Chemicon; Heparitinase and Chondroitinase ABC Protease Free from Seikagaku Corporation; One Solution reagent and anti–phospho-MAPK antibody from Promega; Immobilon-P PVDF membranes from Millipore Corp.; RNeasy Mini Kit from Qiagen; phosphatidylinositol-specific phospholipase C (PI-PLC) from Molecular Probes; magnetic dynabeads from Dynal Biotech from Invitrogen; SulfoLink Coupling Gel from Pierce Biotechnology; anti–ERK-2 antibody, anti–angiopoietin-1 and -2 antibodies, and anti-Tie2 antibody from Santa Cruz Biotechnology Inc.; anti-CD31 rat anti-mouse monoclonal antibody (catalog no. 553370) and anti-VE cadherin antibody from Pharmingen; anti-PCNA monoclonal antibody from Novocastra; Antigen Unmasking Solution from Vector Laboratories; ApopTag in situ apoptosis detection kit from Chemicon; oligonucleotide primers from Applied Biosystems; PANC-1 human pancreatic cancer cells and COS-7 cells from American Type Culture Collection.

    Techniques: Expressing, Transfection, Incubation, Affinity Purification

    Effects of GPC1 antisense expression on GPC1 protein levels. ( A ) Effects of enzymatic treatment on immunoblotting. Sham-transfected cells and GPC1 antisense–expressing cells (clone GAS6) were incubated in the absence or presence of the indicated enzymes and subjected to immunoblotting using affinity-purified rabbit anti-GPC1 antibody as reported in Methods. ( B ) Total cell lysates from sham-transfected and from both antisense-expressing clones as well as the corresponding conditioned media were incubated with heparitinase and chondroitinase ABC and subjected to immunoblotting as described above. Membranes from cell lysates were reprobed with an anti-ERK2 antibody to confirm equivalent loading of lanes. Each panel is representative of 2 distinct experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells

    doi: 10.1172/JCI32412

    Figure Lengend Snippet: Effects of GPC1 antisense expression on GPC1 protein levels. ( A ) Effects of enzymatic treatment on immunoblotting. Sham-transfected cells and GPC1 antisense–expressing cells (clone GAS6) were incubated in the absence or presence of the indicated enzymes and subjected to immunoblotting using affinity-purified rabbit anti-GPC1 antibody as reported in Methods. ( B ) Total cell lysates from sham-transfected and from both antisense-expressing clones as well as the corresponding conditioned media were incubated with heparitinase and chondroitinase ABC and subjected to immunoblotting as described above. Membranes from cell lysates were reprobed with an anti-ERK2 antibody to confirm equivalent loading of lanes. Each panel is representative of 2 distinct experiments.

    Article Snippet: The following materials were purchased from the following companies: DMEM, RPMI 1640, and trypsin-EDTA from Mediatech Inc.; FBS from Omega Scientific Inc.; G418 from GIBCO Laboratories; Noble agar from Difco Laboratories; recombinant human EGF from Chemicon; Heparitinase and Chondroitinase ABC Protease Free from Seikagaku Corporation; One Solution reagent and anti–phospho-MAPK antibody from Promega; Immobilon-P PVDF membranes from Millipore Corp.; RNeasy Mini Kit from Qiagen; phosphatidylinositol-specific phospholipase C (PI-PLC) from Molecular Probes; magnetic dynabeads from Dynal Biotech from Invitrogen; SulfoLink Coupling Gel from Pierce Biotechnology; anti–ERK-2 antibody, anti–angiopoietin-1 and -2 antibodies, and anti-Tie2 antibody from Santa Cruz Biotechnology Inc.; anti-CD31 rat anti-mouse monoclonal antibody (catalog no. 553370) and anti-VE cadherin antibody from Pharmingen; anti-PCNA monoclonal antibody from Novocastra; Antigen Unmasking Solution from Vector Laboratories; ApopTag in situ apoptosis detection kit from Chemicon; oligonucleotide primers from Applied Biosystems; PANC-1 human pancreatic cancer cells and COS-7 cells from American Type Culture Collection.

    Techniques: Expressing, Transfection, Incubation, Affinity Purification, Clone Assay

    The V. fischeri aphrodisiac is a chondroitinase (A) Alignment of the V. fischeri EroS amino acid sequence to diverse bacterial GAG lyases reveals that V. fischeri ). Amino acids with > 50% conservation between sequences are shaded (black shading for identical amino acids and grey shading for similar amino acids. (B) Purified EroS degrades chondroitin sulfate and hyaluronan. EroS was incubated with purified chondroitin sulfate (open circle), hyaluronan (grey hexagon), dermatan sulfate (open square), and heparan sulfate (grey triangle), and GAG lyase activity of EroS was measured by monitoring the abundance of unsaturated oligosaccharide reaction products with an absorbance at 232nm. Chondroitin sulfate and hyaluronan oligosaccharides accumulated rapidly in the presence of EroS, indicating depolymerization, whereas heparan sulfate and dermatan sulfate were not depolymerized by EroS. (C) Alanine substitutions at two predicted catalytic residues in EroS (H278 and Y287) eliminated the protein’s ability to degrade chondroitin sulfate. The chondroitinase activity of either wild type EroS (open circle) or EroS-H278A,Y287A (filled circle) against purified chondroitin sulfate was measured by monitoring the abundance of unsaturated oligosaccharide products with an absorbance at 232nm. (D) The chondroitinase activity of EroS is necessary and sufficient for its function as an aphrodisiac. EroS-H278A,Y287A failed to induce swarming in S. rosetta. P. vulgaris ABC chondroitinase and F. heparinum AC chondroitinase were sufficient to induce swarming at levels similar to EroS, whereas S. hyalurolyticus hyaluronidase failed to induce swarming, indicating that chondroitinase activity is necessary and sufficient for aphrodisiac activity.

    Journal: Cell

    Article Title: Mating in the closest living relatives of animals is induced by a bacterial chondroitinase

    doi: 10.1016/j.cell.2017.08.005

    Figure Lengend Snippet: The V. fischeri aphrodisiac is a chondroitinase (A) Alignment of the V. fischeri EroS amino acid sequence to diverse bacterial GAG lyases reveals that V. fischeri ). Amino acids with > 50% conservation between sequences are shaded (black shading for identical amino acids and grey shading for similar amino acids. (B) Purified EroS degrades chondroitin sulfate and hyaluronan. EroS was incubated with purified chondroitin sulfate (open circle), hyaluronan (grey hexagon), dermatan sulfate (open square), and heparan sulfate (grey triangle), and GAG lyase activity of EroS was measured by monitoring the abundance of unsaturated oligosaccharide reaction products with an absorbance at 232nm. Chondroitin sulfate and hyaluronan oligosaccharides accumulated rapidly in the presence of EroS, indicating depolymerization, whereas heparan sulfate and dermatan sulfate were not depolymerized by EroS. (C) Alanine substitutions at two predicted catalytic residues in EroS (H278 and Y287) eliminated the protein’s ability to degrade chondroitin sulfate. The chondroitinase activity of either wild type EroS (open circle) or EroS-H278A,Y287A (filled circle) against purified chondroitin sulfate was measured by monitoring the abundance of unsaturated oligosaccharide products with an absorbance at 232nm. (D) The chondroitinase activity of EroS is necessary and sufficient for its function as an aphrodisiac. EroS-H278A,Y287A failed to induce swarming in S. rosetta. P. vulgaris ABC chondroitinase and F. heparinum AC chondroitinase were sufficient to induce swarming at levels similar to EroS, whereas S. hyalurolyticus hyaluronidase failed to induce swarming, indicating that chondroitinase activity is necessary and sufficient for aphrodisiac activity.

    Article Snippet: Mating crosses were performed in 2 mL total volumes under the following induction conditions: 5% (V/V) E. pacifica conditioned media (EPCM), 5% (V/V) Vibrio fischeri conditioned media (VFCM), 0.5 nM VF_rGAG lyase, 0.0035 units Chondroitinase ABC (Sigma C3667), and 0.0035 units Chondroitinase AC (Sigma C2780).

    Techniques: Sequencing, Purification, Incubation, Activity Assay

    DKO mice are DS-free. Two-day-old pups were metabolically labeled with 35 S sulfate. Skin decorin CS/DS was extracted and purified (A-C). A) CS/DS chains were analyzed on a size-permeation Superose 6 column. The HS degraded products, which were proportionally increased in the DKO mice, derive from the HS-PGs which co-eluted with decorin in the size permeation column used for PGs separation (see “ Material and methods ”). B) Split products obtained after chondroitinase B treatment were size separated on a Superdex Peptide column and C) the iduronic acid content was calculated. The iduronic acid analysis was conducted in duplicate samples. D) Skin PGs were extracted from E19.5 embryos of different genotypes. Decorin was stained before and after chondroitinase ABC and B treatment.

    Journal: PLoS ONE

    Article Title: Dermatan Sulfate-Free Mice Display Embryological Defects and Are Neonatal Lethal Despite Normal Lymphoid and Non-Lymphoid Organogenesis

    doi: 10.1371/journal.pone.0140279

    Figure Lengend Snippet: DKO mice are DS-free. Two-day-old pups were metabolically labeled with 35 S sulfate. Skin decorin CS/DS was extracted and purified (A-C). A) CS/DS chains were analyzed on a size-permeation Superose 6 column. The HS degraded products, which were proportionally increased in the DKO mice, derive from the HS-PGs which co-eluted with decorin in the size permeation column used for PGs separation (see “ Material and methods ”). B) Split products obtained after chondroitinase B treatment were size separated on a Superdex Peptide column and C) the iduronic acid content was calculated. The iduronic acid analysis was conducted in duplicate samples. D) Skin PGs were extracted from E19.5 embryos of different genotypes. Decorin was stained before and after chondroitinase ABC and B treatment.

    Article Snippet: Quantification and spatial arrangement of IdoA along the chain was analyzed by cleaving 40,000 dpm of CS/DS with 2 mIU of chondroitinase B (Seikagaku) in 20 mM Hepes, pH 7.2, 50 mM NaCl, 4 mM CaCl2 , and 0.1 mg/ml BSA for 90 min.

    Techniques: Mouse Assay, Metabolic Labelling, Labeling, Purification, Staining

    The effect of glycosylated and non-glycosylated biglycan on DRG neurite growth. Graphs showing the mean ± S.D. of number of neurite crossings per DRG when cultured on (A ) ‘biglycan’ at a range of concentrations (2–500 µg/ml) without any enzymatic treatment; (B) ‘biglycan’ at 500 µg/ml following either chondroitinase ABC or chondroitinase AC digestion (both at 250 mU/ml) or in buffer; * P

    Journal: Bioscience Reports

    Article Title: Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

    doi: 10.1042/BSR20160465

    Figure Lengend Snippet: The effect of glycosylated and non-glycosylated biglycan on DRG neurite growth. Graphs showing the mean ± S.D. of number of neurite crossings per DRG when cultured on (A ) ‘biglycan’ at a range of concentrations (2–500 µg/ml) without any enzymatic treatment; (B) ‘biglycan’ at 500 µg/ml following either chondroitinase ABC or chondroitinase AC digestion (both at 250 mU/ml) or in buffer; * P

    Article Snippet: Chondroitinase AC (Seikagaku Corporation, Japan), which was used at concentrations ranging from 2 to 250 mU/ ml, cleaves unsulfated chondroitin, chondroitin-4-sulfate and chondroitin-6-sulfate from the core protein and chondroitinase ABC (also used over a range of 2–250 mU/ml; Sigma–Aldrich) cleaves all of these in addition to dermatan sulfate (DS).

    Techniques: Cell Culture

    Western blot images of aliquots of purified aggrecan isolated from a human intervertebral disc (lanes 2–4), and the commercially sourced ‘biglycan’ (lanes 5–7) and ‘decorin’ (lanes 8–10) following separation on 4–12% Bis-Tris gel PGs in lanes 2, 5 and 8 were undigested whereas those in lanes 3, 6 and 9 and 4, 7 and 10 had been digested with chondroitinase ABC and chondroitinase AC respectively (both 250 mU/ml). Membranes were probed sequentially with monoclonal antibodies to ( A ) over-sulfated KS heptasaccharides (5D4); ( B ) 4-sulfated unsaturated disaccharide CS neo-epitopes (2B6) and (C ) 6-sulfated unsaturated disaccharide CS neo-epitopes (3B3). Lanes 1 and 12 contain molecular weight standards. The expected molecular weight of the intact core protein of both biglycan and decorin is approximately 45 kDa (*). Aggrecan bands were particularly prominent in lane 3 (4C) following chondrointase ABC treatment (arrow), similar bands were seen in the ‘biglycan’ preparation (lane 6, arrow).

    Journal: Bioscience Reports

    Article Title: Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

    doi: 10.1042/BSR20160465

    Figure Lengend Snippet: Western blot images of aliquots of purified aggrecan isolated from a human intervertebral disc (lanes 2–4), and the commercially sourced ‘biglycan’ (lanes 5–7) and ‘decorin’ (lanes 8–10) following separation on 4–12% Bis-Tris gel PGs in lanes 2, 5 and 8 were undigested whereas those in lanes 3, 6 and 9 and 4, 7 and 10 had been digested with chondroitinase ABC and chondroitinase AC respectively (both 250 mU/ml). Membranes were probed sequentially with monoclonal antibodies to ( A ) over-sulfated KS heptasaccharides (5D4); ( B ) 4-sulfated unsaturated disaccharide CS neo-epitopes (2B6) and (C ) 6-sulfated unsaturated disaccharide CS neo-epitopes (3B3). Lanes 1 and 12 contain molecular weight standards. The expected molecular weight of the intact core protein of both biglycan and decorin is approximately 45 kDa (*). Aggrecan bands were particularly prominent in lane 3 (4C) following chondrointase ABC treatment (arrow), similar bands were seen in the ‘biglycan’ preparation (lane 6, arrow).

    Article Snippet: Chondroitinase AC (Seikagaku Corporation, Japan), which was used at concentrations ranging from 2 to 250 mU/ ml, cleaves unsulfated chondroitin, chondroitin-4-sulfate and chondroitin-6-sulfate from the core protein and chondroitinase ABC (also used over a range of 2–250 mU/ml; Sigma–Aldrich) cleaves all of these in addition to dermatan sulfate (DS).

    Techniques: Western Blot, Purification, Isolation, Molecular Weight

    Image of one of the 12.5% SDS-PAGE stained with Coomassie Blue following electrophoretic separation of the commercially sourced ‘decorin’ (1st batches (1) and 2nd batch (2)) and ‘biglycan’ after enzymatic digestion with chondroitinase ABC, keratanase and keratanase II (removing all CS, DS and KS GAG chains) Protein slices as outlined (1–8) were dissected from each lane and then prepared for MS analyses.

    Journal: Bioscience Reports

    Article Title: Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

    doi: 10.1042/BSR20160465

    Figure Lengend Snippet: Image of one of the 12.5% SDS-PAGE stained with Coomassie Blue following electrophoretic separation of the commercially sourced ‘decorin’ (1st batches (1) and 2nd batch (2)) and ‘biglycan’ after enzymatic digestion with chondroitinase ABC, keratanase and keratanase II (removing all CS, DS and KS GAG chains) Protein slices as outlined (1–8) were dissected from each lane and then prepared for MS analyses.

    Article Snippet: Chondroitinase AC (Seikagaku Corporation, Japan), which was used at concentrations ranging from 2 to 250 mU/ ml, cleaves unsulfated chondroitin, chondroitin-4-sulfate and chondroitin-6-sulfate from the core protein and chondroitinase ABC (also used over a range of 2–250 mU/ml; Sigma–Aldrich) cleaves all of these in addition to dermatan sulfate (DS).

    Techniques: SDS Page, Staining, Mass Spectrometry

    Western blot images of aliquots of the commercially sourced ‘decorin’ and ‘biglycan’ and purified human aggrecan following separation on 4–12% Bis-Tris gels The PGs in ( A )–( C ) had been digested with chondroitinase ABC, keratanase and keratanase II (removing CS, DS and KS GAG chains). Lanes 1 and 3 of (A)–(C) contain different batches of ‘decorin’, lane 4 contains ‘biglycan’ and lane 2 contains the molecular weight standards. The expected molecular weight of the intact core protein of both biglycan and decorin is approximately 45 kDa (*). The membranes were probed with antibodies to (A) biglycan (PR-85); (B) decorin (DS-1) and ( C ) aggrecan (6B4). For the membranes in ( D )–( F ), lane 1 contains the molecular weight standards; lanes 2 and 3 contain ‘biglycan’ without and with prior enzymatic digestion with chondroitinase ABC respectively and in lane 4 is purified human aggrecan without prior digestion. These membranes were probed with ( D) fibromodulin; (E) 1B4, which detects less-sulfated KS and (F) lumican.

    Journal: Bioscience Reports

    Article Title: Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

    doi: 10.1042/BSR20160465

    Figure Lengend Snippet: Western blot images of aliquots of the commercially sourced ‘decorin’ and ‘biglycan’ and purified human aggrecan following separation on 4–12% Bis-Tris gels The PGs in ( A )–( C ) had been digested with chondroitinase ABC, keratanase and keratanase II (removing CS, DS and KS GAG chains). Lanes 1 and 3 of (A)–(C) contain different batches of ‘decorin’, lane 4 contains ‘biglycan’ and lane 2 contains the molecular weight standards. The expected molecular weight of the intact core protein of both biglycan and decorin is approximately 45 kDa (*). The membranes were probed with antibodies to (A) biglycan (PR-85); (B) decorin (DS-1) and ( C ) aggrecan (6B4). For the membranes in ( D )–( F ), lane 1 contains the molecular weight standards; lanes 2 and 3 contain ‘biglycan’ without and with prior enzymatic digestion with chondroitinase ABC respectively and in lane 4 is purified human aggrecan without prior digestion. These membranes were probed with ( D) fibromodulin; (E) 1B4, which detects less-sulfated KS and (F) lumican.

    Article Snippet: Chondroitinase AC (Seikagaku Corporation, Japan), which was used at concentrations ranging from 2 to 250 mU/ ml, cleaves unsulfated chondroitin, chondroitin-4-sulfate and chondroitin-6-sulfate from the core protein and chondroitinase ABC (also used over a range of 2–250 mU/ml; Sigma–Aldrich) cleaves all of these in addition to dermatan sulfate (DS).

    Techniques: Western Blot, Purification, Molecular Weight

    The effect of glycosylated and non-glycosylated decorin on DRG neurite growth. Graphs showing the mean ± S.D. of number of neurite crossings per DRG when cultured on ( A ) ‘decorin’ at a range of concentrations (10–1000 µg/ml) without any enzymatic treatment; ( B ) ‘decorin’ at 500 µg/ml following chondroitinase ABC and chondroitinase AC treatment (both at 250 mU/ml) or in buffer; ** P

    Journal: Bioscience Reports

    Article Title: Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

    doi: 10.1042/BSR20160465

    Figure Lengend Snippet: The effect of glycosylated and non-glycosylated decorin on DRG neurite growth. Graphs showing the mean ± S.D. of number of neurite crossings per DRG when cultured on ( A ) ‘decorin’ at a range of concentrations (10–1000 µg/ml) without any enzymatic treatment; ( B ) ‘decorin’ at 500 µg/ml following chondroitinase ABC and chondroitinase AC treatment (both at 250 mU/ml) or in buffer; ** P

    Article Snippet: Chondroitinase AC (Seikagaku Corporation, Japan), which was used at concentrations ranging from 2 to 250 mU/ ml, cleaves unsulfated chondroitin, chondroitin-4-sulfate and chondroitin-6-sulfate from the core protein and chondroitinase ABC (also used over a range of 2–250 mU/ml; Sigma–Aldrich) cleaves all of these in addition to dermatan sulfate (DS).

    Techniques: Cell Culture

    ( A ) NRP1 is GAG modified on a single Ser 612 residue. CASMCs were transfected with adenoviral vectors encoding WT or S612A mutant NRP1. NRP1 S612A is not GAG modified. ( B ) Multiple alignments of NRP1 from different species. Ser 612 is highly conserved among vertebrates. ( C ) NRP2, an NRP family member, is not GAG modified. ( D ) Design of siRNA and adenovirus constructs. Ser 612 exists in the bridge region between the b1b2 and MAM domains. ( E ) Replacement of Ser 612 by Ala 612 of NRP1 did not change binding to VEGF. Cos7 cells were transfected with either NRP1 WT′ or S612A expression vector and preincubated with heparitinase (1.5 mU/ml), heparinase (1.5 mU/ml), and chondroitinase (20 mU/ml) in the culture medium at 37°C for 2 h to make NRP1 non-GAG form. After incubation with 125 I-labeled VEGF for 30 min at room temperature, cell lysates were immunoprecipitated by anti-NRP1 antibody, and the bound radioactivity was quantitated using a gamma counter. Data are from three independent experiments. For panel E, error bars represent s.e.

    Journal: The EMBO Journal

    Article Title: Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling

    doi: 10.1038/sj.emboj.7601188

    Figure Lengend Snippet: ( A ) NRP1 is GAG modified on a single Ser 612 residue. CASMCs were transfected with adenoviral vectors encoding WT or S612A mutant NRP1. NRP1 S612A is not GAG modified. ( B ) Multiple alignments of NRP1 from different species. Ser 612 is highly conserved among vertebrates. ( C ) NRP2, an NRP family member, is not GAG modified. ( D ) Design of siRNA and adenovirus constructs. Ser 612 exists in the bridge region between the b1b2 and MAM domains. ( E ) Replacement of Ser 612 by Ala 612 of NRP1 did not change binding to VEGF. Cos7 cells were transfected with either NRP1 WT′ or S612A expression vector and preincubated with heparitinase (1.5 mU/ml), heparinase (1.5 mU/ml), and chondroitinase (20 mU/ml) in the culture medium at 37°C for 2 h to make NRP1 non-GAG form. After incubation with 125 I-labeled VEGF for 30 min at room temperature, cell lysates were immunoprecipitated by anti-NRP1 antibody, and the bound radioactivity was quantitated using a gamma counter. Data are from three independent experiments. For panel E, error bars represent s.e.

    Article Snippet: Heparitinase, heparinase, and chondroitinase were purchased from Seikagaku Corp.

    Techniques: Modification, Transfection, Mutagenesis, Construct, Binding Assay, Expressing, Plasmid Preparation, Incubation, Labeling, Immunoprecipitation, Radioactivity

    A substantial fraction of cellular NRP1 is proteoglycan, composed of either HS or CS. ( A ) 125 I-labeled VEGF is crosslinked to different proteins in ECs and SMCs. Arrow indicates VEGF-binding protein specifically seen in SMCs. CASMC: coronary artery smooth muscle cell; BSMC: bronchial smooth muscle cell; HUVEC: human umbilical vein endothelial cell. ( B ) Western blots of exogenously expressed NRP1 in either CASMCs or HUVECs. Adenovirus encoding FLAG-tagged NRP1 was transfected 2 days before analysis at the indicated MOI. LacZ-encoding adenovirus was used as a control. ( C ) The high molecular weight band was not simply a covalently linked homodimer of NRP1. Only FLAG-tagged NRP1 or both FLAG-tagged and V5-tagged NRP1 were transfected in CASMCs, and the cell lysates were immunoprecipitated and detected by the indicated antibody. ( D ) Endogenous NRP1 was modified by GAG chain addition in both SMCs and ECs. The upper band in CASMC immunoprecipitates disappeared following treatment with both HSase and CSase. HUVEC-expressed NRP1 is also GAG modified. The band intensity was analyzed and the proportion of each glycanated form of NRP1 was determined. Data are from three separate experiments. HSase: heparitinase; CSase: chondroitinase.

    Journal: The EMBO Journal

    Article Title: Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling

    doi: 10.1038/sj.emboj.7601188

    Figure Lengend Snippet: A substantial fraction of cellular NRP1 is proteoglycan, composed of either HS or CS. ( A ) 125 I-labeled VEGF is crosslinked to different proteins in ECs and SMCs. Arrow indicates VEGF-binding protein specifically seen in SMCs. CASMC: coronary artery smooth muscle cell; BSMC: bronchial smooth muscle cell; HUVEC: human umbilical vein endothelial cell. ( B ) Western blots of exogenously expressed NRP1 in either CASMCs or HUVECs. Adenovirus encoding FLAG-tagged NRP1 was transfected 2 days before analysis at the indicated MOI. LacZ-encoding adenovirus was used as a control. ( C ) The high molecular weight band was not simply a covalently linked homodimer of NRP1. Only FLAG-tagged NRP1 or both FLAG-tagged and V5-tagged NRP1 were transfected in CASMCs, and the cell lysates were immunoprecipitated and detected by the indicated antibody. ( D ) Endogenous NRP1 was modified by GAG chain addition in both SMCs and ECs. The upper band in CASMC immunoprecipitates disappeared following treatment with both HSase and CSase. HUVEC-expressed NRP1 is also GAG modified. The band intensity was analyzed and the proportion of each glycanated form of NRP1 was determined. Data are from three separate experiments. HSase: heparitinase; CSase: chondroitinase.

    Article Snippet: Heparitinase, heparinase, and chondroitinase were purchased from Seikagaku Corp.

    Techniques: Labeling, Binding Assay, Western Blot, Transfection, Molecular Weight, Immunoprecipitation, Modification

    GAG modifications differentially affect NRP1 function in SMCs and ECs. ( A ) Experimental replacement of NRP1 in SMCs and ECs. After transfection with both N-G siRNA and adenoviral constructs, endogenous NRP1 was successfully replaced with either the glycanated form (NRP1 WT′) or non-glycanated form (NRP1 S612A) of NRP1. Tubulin was used as a loading control. ( B ) Addition of GAG to NRP1 enhances binding to VEGF in both types of cells. Two days after NRP1 replacement, cell lysates were immunoprecipitated with anti-FLAG antibody after incubation with 125 I-labeled VEGF (25 ng/ml) for 40 min at room temperature, and bound radioactivity was quantitated using a gamma counter. Heparitinase and chondroitinase treatment with these immunoprecipitates could not entirely eliminate the enhancement of VEGF binding. Data are from three independent experiments. ( C ) VEGF (50 ng/ml) induced greater cell migration in SMCs expressing non-modified NRP1 S612A than those expressing NRP1 WT′. Migrated cells were quantified by counting cells in three random high-power fields (HPF, × 200). Similar results were obtained from additional two independent experiments. ( D ) VEGF (50 ng/ml) increased cell viability in ECs expressing NRP1 WT′ to a greater extent than in ECs expressing NRP1 S612A. Data are from three independent experiments. For panels B–D, error bars represent s.e. * P

    Journal: The EMBO Journal

    Article Title: Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling

    doi: 10.1038/sj.emboj.7601188

    Figure Lengend Snippet: GAG modifications differentially affect NRP1 function in SMCs and ECs. ( A ) Experimental replacement of NRP1 in SMCs and ECs. After transfection with both N-G siRNA and adenoviral constructs, endogenous NRP1 was successfully replaced with either the glycanated form (NRP1 WT′) or non-glycanated form (NRP1 S612A) of NRP1. Tubulin was used as a loading control. ( B ) Addition of GAG to NRP1 enhances binding to VEGF in both types of cells. Two days after NRP1 replacement, cell lysates were immunoprecipitated with anti-FLAG antibody after incubation with 125 I-labeled VEGF (25 ng/ml) for 40 min at room temperature, and bound radioactivity was quantitated using a gamma counter. Heparitinase and chondroitinase treatment with these immunoprecipitates could not entirely eliminate the enhancement of VEGF binding. Data are from three independent experiments. ( C ) VEGF (50 ng/ml) induced greater cell migration in SMCs expressing non-modified NRP1 S612A than those expressing NRP1 WT′. Migrated cells were quantified by counting cells in three random high-power fields (HPF, × 200). Similar results were obtained from additional two independent experiments. ( D ) VEGF (50 ng/ml) increased cell viability in ECs expressing NRP1 WT′ to a greater extent than in ECs expressing NRP1 S612A. Data are from three independent experiments. For panels B–D, error bars represent s.e. * P

    Article Snippet: Heparitinase, heparinase, and chondroitinase were purchased from Seikagaku Corp.

    Techniques: Transfection, Construct, Binding Assay, Immunoprecipitation, Incubation, Labeling, Radioactivity, Migration, Expressing, Modification

    NREs common to XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. A–C , average HCD-MS 2 spectra of the NRE precursor ions at m/z 380.00 [1−] ( A ), 476.07 [1−] ( B ), and 490.09 [1−] ( C ) observed in chondroitinase ABC-degraded XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. D , incorporation of [ 3 H]methyl in XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells expressed as radioactivity in dpm per μg of GAGs. E , average HCD-MS 2 spectrum of the NRE precursor ion at m/z 556.01 [1−] observed in chondroitinase AC-I– and -II–degraded XylNap-primed GAGs from HCC70 cells. The nomenclature of the fragment ion at m/z ).

    Journal: The Journal of Biological Chemistry

    Article Title: LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

    doi: 10.1074/jbc.RA118.002971

    Figure Lengend Snippet: NREs common to XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. A–C , average HCD-MS 2 spectra of the NRE precursor ions at m/z 380.00 [1−] ( A ), 476.07 [1−] ( B ), and 490.09 [1−] ( C ) observed in chondroitinase ABC-degraded XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. D , incorporation of [ 3 H]methyl in XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells expressed as radioactivity in dpm per μg of GAGs. E , average HCD-MS 2 spectrum of the NRE precursor ion at m/z 556.01 [1−] observed in chondroitinase AC-I– and -II–degraded XylNap-primed GAGs from HCC70 cells. The nomenclature of the fragment ion at m/z ).

    Article Snippet: Approximately 10 μg of XylNap- and XylNap- d 7 -primed GAGs were degraded using either chondroitinase ABC (EC 4.2.2.20) (Seikagaku), chondroitinase AC-I and -II (EC 4.2.2.5) (Seikagaku), chondroitinase B (EC 4.2.2.19) (R & D Systems), or heparinase II (no EC number) and heparinase III (EC 4.2.2.8) (from Flavobacterium heparinum overexpressed in Escherichia coli , a gift from Prof. Jian Liu, University of North Carolina).

    Techniques: Mass Spectrometry, Radioactivity

    Relative proportions of CS/DS and HS and disaccharide composition of XylNap-primed CS/DS from HCC70 cells and CCD-1095Sk cells. A , relative proportions of GlcUA in CS/DS (CS/DS GlcUA ), IdoUA in CS/DS distributed as alternating or single IdoUA-containing units (CS/DS IdoUA_Alt/single ), IdoUA in CS/DS distributed as blocks (CS/DS IdoUA_ChB ), and HS of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells, respectively. B and C , disaccharide composition of XylNap-primed GAGs from HCC70 cells ( B ) and CCD-1095Sk cells ( C ) after depolymerization with chondroitinase ABC (total), chondroitinase AC-I and -II ( ChAC ), chondroitinase B ( ChB ), and the remaining disaccharides degraded by chondroitinase ABC but not by chondroitinase AC-I and -II and chondroitinase B ( Ch ( ABC-AC-B .

    Journal: The Journal of Biological Chemistry

    Article Title: LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

    doi: 10.1074/jbc.RA118.002971

    Figure Lengend Snippet: Relative proportions of CS/DS and HS and disaccharide composition of XylNap-primed CS/DS from HCC70 cells and CCD-1095Sk cells. A , relative proportions of GlcUA in CS/DS (CS/DS GlcUA ), IdoUA in CS/DS distributed as alternating or single IdoUA-containing units (CS/DS IdoUA_Alt/single ), IdoUA in CS/DS distributed as blocks (CS/DS IdoUA_ChB ), and HS of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells, respectively. B and C , disaccharide composition of XylNap-primed GAGs from HCC70 cells ( B ) and CCD-1095Sk cells ( C ) after depolymerization with chondroitinase ABC (total), chondroitinase AC-I and -II ( ChAC ), chondroitinase B ( ChB ), and the remaining disaccharides degraded by chondroitinase ABC but not by chondroitinase AC-I and -II and chondroitinase B ( Ch ( ABC-AC-B .

    Article Snippet: Approximately 10 μg of XylNap- and XylNap- d 7 -primed GAGs were degraded using either chondroitinase ABC (EC 4.2.2.20) (Seikagaku), chondroitinase AC-I and -II (EC 4.2.2.5) (Seikagaku), chondroitinase B (EC 4.2.2.19) (R & D Systems), or heparinase II (no EC number) and heparinase III (EC 4.2.2.8) (from Flavobacterium heparinum overexpressed in Escherichia coli , a gift from Prof. Jian Liu, University of North Carolina).

    Techniques:

    Growth of HCC70 cells in the presence of enzymatically degraded XylNap-primed CS/DS from HCC70 cells. The growth of HCC70 cells after 96 h of treatment with XylNap-primed GAGs degraded with heparinase II and III ( Hep ; black ), heparinase II and III and chondroitinase ABC ( Hep + ChABC ; gray ), heparinase II and III and chondroitinase AC-I and -II ( Hep + ChAC ; blue ), or heparinase II and III and chondroitinase B ( Hep + ChB ; white ). The concentrations of the GAGs administered to the cells were de facto lower than the indicated concentrations, as the indicated concentrations correspond to the concentrations of the GAGs before enzymatic degradation. The data points are the means ± S.D., in which n = 3. UT , untreated.

    Journal: The Journal of Biological Chemistry

    Article Title: LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

    doi: 10.1074/jbc.RA118.002971

    Figure Lengend Snippet: Growth of HCC70 cells in the presence of enzymatically degraded XylNap-primed CS/DS from HCC70 cells. The growth of HCC70 cells after 96 h of treatment with XylNap-primed GAGs degraded with heparinase II and III ( Hep ; black ), heparinase II and III and chondroitinase ABC ( Hep + ChABC ; gray ), heparinase II and III and chondroitinase AC-I and -II ( Hep + ChAC ; blue ), or heparinase II and III and chondroitinase B ( Hep + ChB ; white ). The concentrations of the GAGs administered to the cells were de facto lower than the indicated concentrations, as the indicated concentrations correspond to the concentrations of the GAGs before enzymatic degradation. The data points are the means ± S.D., in which n = 3. UT , untreated.

    Article Snippet: Approximately 10 μg of XylNap- and XylNap- d 7 -primed GAGs were degraded using either chondroitinase ABC (EC 4.2.2.20) (Seikagaku), chondroitinase AC-I and -II (EC 4.2.2.5) (Seikagaku), chondroitinase B (EC 4.2.2.19) (R & D Systems), or heparinase II (no EC number) and heparinase III (EC 4.2.2.8) (from Flavobacterium heparinum overexpressed in Escherichia coli , a gift from Prof. Jian Liu, University of North Carolina).

    Techniques:

    Base peak chromatograms of chondroitinase AC-I– and -II–degraded and chondroitinase B–degraded XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells. A and B , degradation products of XylNap-primed GAGs from HCC70 cells ( A ) and CCD-1095Sk cells ( B ) generated after chondroitinase AC-I and -II ( gray ) or chondroitinase B ( pink ) treatment, containing disaccharides (dp2), oligosaccharides (dp4 and dp6), and linkage regions ( L4 to L10 ) carrying one to three sulfate groups ( S1–S3 ) and/or one Neu5Ac ( SA1 ).

    Journal: The Journal of Biological Chemistry

    Article Title: LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

    doi: 10.1074/jbc.RA118.002971

    Figure Lengend Snippet: Base peak chromatograms of chondroitinase AC-I– and -II–degraded and chondroitinase B–degraded XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells. A and B , degradation products of XylNap-primed GAGs from HCC70 cells ( A ) and CCD-1095Sk cells ( B ) generated after chondroitinase AC-I and -II ( gray ) or chondroitinase B ( pink ) treatment, containing disaccharides (dp2), oligosaccharides (dp4 and dp6), and linkage regions ( L4 to L10 ) carrying one to three sulfate groups ( S1–S3 ) and/or one Neu5Ac ( SA1 ).

    Article Snippet: Approximately 10 μg of XylNap- and XylNap- d 7 -primed GAGs were degraded using either chondroitinase ABC (EC 4.2.2.20) (Seikagaku), chondroitinase AC-I and -II (EC 4.2.2.5) (Seikagaku), chondroitinase B (EC 4.2.2.19) (R & D Systems), or heparinase II (no EC number) and heparinase III (EC 4.2.2.8) (from Flavobacterium heparinum overexpressed in Escherichia coli , a gift from Prof. Jian Liu, University of North Carolina).

    Techniques: Generated

    Total ion chromatograms of enzymatically degraded XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells and MS 1 of intact XylNap-primed CS/DS. A–F , degradation products of XylNap-primed GAGs from HCC70 cells ( A , C , and E ) and CCD-1095Sk cells ( B, D, and F ) generated after heparinase II and III ( A and B ), chondroitinase AC-I and -II ( C and D ), or chondroitinase B ( E and F ) treatment. G–I , full MS spectra of the peaks in A eluting at 41.81–42.40 min ( G ), 43.61–44.11 min ( H ), and 45.99–46.98 min ( I ), corresponding to intact XylNap-primed CS/DS ( L11 to L19 ) carrying five to seven sulfate groups ( S5–S7 ) and/or one Neu5Ac ( SA1 ). DBA, dibutylamine.

    Journal: The Journal of Biological Chemistry

    Article Title: LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

    doi: 10.1074/jbc.RA118.002971

    Figure Lengend Snippet: Total ion chromatograms of enzymatically degraded XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells and MS 1 of intact XylNap-primed CS/DS. A–F , degradation products of XylNap-primed GAGs from HCC70 cells ( A , C , and E ) and CCD-1095Sk cells ( B, D, and F ) generated after heparinase II and III ( A and B ), chondroitinase AC-I and -II ( C and D ), or chondroitinase B ( E and F ) treatment. G–I , full MS spectra of the peaks in A eluting at 41.81–42.40 min ( G ), 43.61–44.11 min ( H ), and 45.99–46.98 min ( I ), corresponding to intact XylNap-primed CS/DS ( L11 to L19 ) carrying five to seven sulfate groups ( S5–S7 ) and/or one Neu5Ac ( SA1 ). DBA, dibutylamine.

    Article Snippet: Approximately 10 μg of XylNap- and XylNap- d 7 -primed GAGs were degraded using either chondroitinase ABC (EC 4.2.2.20) (Seikagaku), chondroitinase AC-I and -II (EC 4.2.2.5) (Seikagaku), chondroitinase B (EC 4.2.2.19) (R & D Systems), or heparinase II (no EC number) and heparinase III (EC 4.2.2.8) (from Flavobacterium heparinum overexpressed in Escherichia coli , a gift from Prof. Jian Liu, University of North Carolina).

    Techniques: Mass Spectrometry, Generated

    Proposed distribution of GlcUA and IdoUA in CS/DS from HCC70 cells and CCD-1095Sk cells primed on XylNap. A and B , proposed structures of XylNap-primed CS/DS from HCC70 cells ( A ) and CCD-1095Sk cells ( B ). The distribution of IdoUA and GlcUA was based on the MS data of the main products generated after chondroitinase AC-I and -II and chondroitinase B degradation. Two parallel lines indicate that the chain either ends or continues with the following polysaccharide. B ), corresponding to an average length of 30 disaccharides. Sulfation was not taken into consideration.

    Journal: The Journal of Biological Chemistry

    Article Title: LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

    doi: 10.1074/jbc.RA118.002971

    Figure Lengend Snippet: Proposed distribution of GlcUA and IdoUA in CS/DS from HCC70 cells and CCD-1095Sk cells primed on XylNap. A and B , proposed structures of XylNap-primed CS/DS from HCC70 cells ( A ) and CCD-1095Sk cells ( B ). The distribution of IdoUA and GlcUA was based on the MS data of the main products generated after chondroitinase AC-I and -II and chondroitinase B degradation. Two parallel lines indicate that the chain either ends or continues with the following polysaccharide. B ), corresponding to an average length of 30 disaccharides. Sulfation was not taken into consideration.

    Article Snippet: Approximately 10 μg of XylNap- and XylNap- d 7 -primed GAGs were degraded using either chondroitinase ABC (EC 4.2.2.20) (Seikagaku), chondroitinase AC-I and -II (EC 4.2.2.5) (Seikagaku), chondroitinase B (EC 4.2.2.19) (R & D Systems), or heparinase II (no EC number) and heparinase III (EC 4.2.2.8) (from Flavobacterium heparinum overexpressed in Escherichia coli , a gift from Prof. Jian Liu, University of North Carolina).

    Techniques: Mass Spectrometry, Generated

    CPG-1 through -6 behave as CSPGs in COS-7 cells. CPG-1 through -6 were expressed as Myc-tagged recombinant proteins in COS-7 cells. Proteoglycans from conditioned media were purified by anion-exchange chromatography (see Materials and Methods). Samples were digested with chondroitinase ABC (+) or left untreated (−), separated by SDS-PAGE, and Western blotted with an anti-Myc mAb.

    Journal: The Journal of Cell Biology

    Article Title: Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2

    doi: 10.1083/jcb.200603003

    Figure Lengend Snippet: CPG-1 through -6 behave as CSPGs in COS-7 cells. CPG-1 through -6 were expressed as Myc-tagged recombinant proteins in COS-7 cells. Proteoglycans from conditioned media were purified by anion-exchange chromatography (see Materials and Methods). Samples were digested with chondroitinase ABC (+) or left untreated (−), separated by SDS-PAGE, and Western blotted with an anti-Myc mAb.

    Article Snippet: Western blotting Proteoglycan samples (10 μL) were digested with 10 mU chondroitinase ABC (Seikagaku) and/or 2.5 mU heparin lyase II (Sigma-Aldrich) for 3–5 h at 37°C.

    Techniques: Recombinant, Purification, Chromatography, SDS Page, Western Blot

    C. elegans CPGs identified by BEMAD/MUDPIT. CPGs were purified from worm extracts and identified by mass spectrometry analysis. Nine independent purifications resulted in identification of nine CPGs, most of which were identified in multiple runs. The predicted masses are based on amino acid sequence, and the apparent masses are based on SDS-PAGE migration of recombinant proteins expressed in COS-7 cells after digestion with chondroitinase ABC and reduction. Putative glycosylation sites (short vertical lines) consist of Ser-Gly dipeptides flanked by one or more acidic amino acids. Identified sites (long vertical lines) represent serine residues modified with DTT by the BEMAD method (see Materials and methods). Genes enriched for germline expression were identified by Reinke et al. (2000) . Schematic drawings of each CPG are shown. Gray circles indicate signal peptides, black ovals are peritrophin-A chitin binding domains, diagonally hatched boxes identify C-type lectin domains, and dotted lines below the protein indicate peptide coverage discovered by mass spectrometry.

    Journal: The Journal of Cell Biology

    Article Title: Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2

    doi: 10.1083/jcb.200603003

    Figure Lengend Snippet: C. elegans CPGs identified by BEMAD/MUDPIT. CPGs were purified from worm extracts and identified by mass spectrometry analysis. Nine independent purifications resulted in identification of nine CPGs, most of which were identified in multiple runs. The predicted masses are based on amino acid sequence, and the apparent masses are based on SDS-PAGE migration of recombinant proteins expressed in COS-7 cells after digestion with chondroitinase ABC and reduction. Putative glycosylation sites (short vertical lines) consist of Ser-Gly dipeptides flanked by one or more acidic amino acids. Identified sites (long vertical lines) represent serine residues modified with DTT by the BEMAD method (see Materials and methods). Genes enriched for germline expression were identified by Reinke et al. (2000) . Schematic drawings of each CPG are shown. Gray circles indicate signal peptides, black ovals are peritrophin-A chitin binding domains, diagonally hatched boxes identify C-type lectin domains, and dotted lines below the protein indicate peptide coverage discovered by mass spectrometry.

    Article Snippet: Western blotting Proteoglycan samples (10 μL) were digested with 10 mU chondroitinase ABC (Seikagaku) and/or 2.5 mU heparin lyase II (Sigma-Aldrich) for 3–5 h at 37°C.

    Techniques: Purification, Mass Spectrometry, Sequencing, SDS Page, Migration, Recombinant, Modification, Expressing, Binding Assay

    C. elegans express multiple CPGs. (A) Glycopeptides were treated with alkali to produce free glycans, which were then analyzed by gel filtration HPLC (see Materials and methods). Untreated sample eluted as two peaks (indicated by squares), whereas after chondroitinase ABC all the material eluted as disaccharides (indicated by circles). V i , elution position of [1- 3 H]glucose; V o , elution position of blue dextran. Shark cartilage chondroitin sulfate (∼20 kD) was run as a standard. (B) Crude extracts of adult worms (lanes 1–3) or embryos (lane 4) were digested with chondroitinase ABC (lanes 2–4) and separated by SDS-PAGE. Western blotting was performed with mAb 1-B-5, which recognizes a neoepitope generated by chondroitinase digestion. The undigested control (lane 1) demonstrates the requirement of the antibody for chondroitin stubs. Lanes 2 and 4, 10-μg protein; lanes 1 and 3, 40-μg protein. The carats indicate the position of protein bands that arose only after chondroitinase ABC digestion.

    Journal: The Journal of Cell Biology

    Article Title: Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2

    doi: 10.1083/jcb.200603003

    Figure Lengend Snippet: C. elegans express multiple CPGs. (A) Glycopeptides were treated with alkali to produce free glycans, which were then analyzed by gel filtration HPLC (see Materials and methods). Untreated sample eluted as two peaks (indicated by squares), whereas after chondroitinase ABC all the material eluted as disaccharides (indicated by circles). V i , elution position of [1- 3 H]glucose; V o , elution position of blue dextran. Shark cartilage chondroitin sulfate (∼20 kD) was run as a standard. (B) Crude extracts of adult worms (lanes 1–3) or embryos (lane 4) were digested with chondroitinase ABC (lanes 2–4) and separated by SDS-PAGE. Western blotting was performed with mAb 1-B-5, which recognizes a neoepitope generated by chondroitinase digestion. The undigested control (lane 1) demonstrates the requirement of the antibody for chondroitin stubs. Lanes 2 and 4, 10-μg protein; lanes 1 and 3, 40-μg protein. The carats indicate the position of protein bands that arose only after chondroitinase ABC digestion.

    Article Snippet: Western blotting Proteoglycan samples (10 μL) were digested with 10 mU chondroitinase ABC (Seikagaku) and/or 2.5 mU heparin lyase II (Sigma-Aldrich) for 3–5 h at 37°C.

    Techniques: Filtration, High Performance Liquid Chromatography, SDS Page, Western Blot, Generated

    CPG-1 and -2 bind chitin and exist as CPGs in vivo. (A) Proteoglycan extracts of vector and RNAi-treated animals were digested with chondroitinase ABC, analyzed by SDS-PAGE, and Western blotted with the 1B5 mAb that recognizes the chondroitin stub remaining after enzyme digestion. cpg-2(RNAi) reduced the bands at 120, ∼80, and ∼45 kD (arrowheads). (B) 20 μg of total worm extract (input) was incubated with chitin beads. Four bands specifically bound chitin (arrowheads), but the 60-kD band did not. (C) Proteoglycan extracts from RNAi-treated animals were enriched by affinity chromatography on chitin beads. cpg-1(RNAi) reduced the band at ∼150 kD (open arrowhead), whereas cpg-2(RNAi) reduced the bands at 120, ∼80, and 45 kD (filled arrowheads).

    Journal: The Journal of Cell Biology

    Article Title: Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2

    doi: 10.1083/jcb.200603003

    Figure Lengend Snippet: CPG-1 and -2 bind chitin and exist as CPGs in vivo. (A) Proteoglycan extracts of vector and RNAi-treated animals were digested with chondroitinase ABC, analyzed by SDS-PAGE, and Western blotted with the 1B5 mAb that recognizes the chondroitin stub remaining after enzyme digestion. cpg-2(RNAi) reduced the bands at 120, ∼80, and ∼45 kD (arrowheads). (B) 20 μg of total worm extract (input) was incubated with chitin beads. Four bands specifically bound chitin (arrowheads), but the 60-kD band did not. (C) Proteoglycan extracts from RNAi-treated animals were enriched by affinity chromatography on chitin beads. cpg-1(RNAi) reduced the band at ∼150 kD (open arrowhead), whereas cpg-2(RNAi) reduced the bands at 120, ∼80, and 45 kD (filled arrowheads).

    Article Snippet: Western blotting Proteoglycan samples (10 μL) were digested with 10 mU chondroitinase ABC (Seikagaku) and/or 2.5 mU heparin lyase II (Sigma-Aldrich) for 3–5 h at 37°C.

    Techniques: In Vivo, Plasmid Preparation, SDS Page, Western Blot, Incubation, Affinity Chromatography

    Pharmacological knockdown of other glycosaminoglycan (GAG) family members does not affect Aβ 1-40 -mediated ROS production in VSMC. Primary human cerebral VSMC were pre-treated with chondroitinase B (10 -1 IU/mL; selectively degrades dermatin sulfate; panel a ), chondroitinase AC (10 -1 IU/mL; selectively degrades chondroitin sulfate; panel a ), or varying concentrations of the sulfation inhibitor sodium chlorate (5-50 mM; panel b ) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM), and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. Results are representative of 3 independent experiments performed in triplicate. *p

    Journal: Molecular Neurodegeneration

    Article Title: Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells

    doi: 10.1186/s13024-016-0073-8

    Figure Lengend Snippet: Pharmacological knockdown of other glycosaminoglycan (GAG) family members does not affect Aβ 1-40 -mediated ROS production in VSMC. Primary human cerebral VSMC were pre-treated with chondroitinase B (10 -1 IU/mL; selectively degrades dermatin sulfate; panel a ), chondroitinase AC (10 -1 IU/mL; selectively degrades chondroitin sulfate; panel a ), or varying concentrations of the sulfation inhibitor sodium chlorate (5-50 mM; panel b ) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM), and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. Results are representative of 3 independent experiments performed in triplicate. *p

    Article Snippet: Chondroitinase B and AC were purchased from IBEX Technologies Inc. (Montreal, Quebec, Canada).

    Techniques: Concentration Assay, Fluorescence

    VP16-KLF7 and Lenti-chondroitinase increase the proximity of injured sensory axons to sites of spinal injury but do not promote regeneration beyond the injury Sensory neurons in adult mice were transduced by lumbar puncture delivery of AAV-EBFP-2A-mCherry or AAV-VP16-2A-mCherry by lumbar puncture, and received cervical dorsal hemisections accompanied by either control injections or spinal injections of Lenti-Chase. Four weeks later ascending sensory axons were labeled by injection of Dextran-Alexafluor 488 injections to the sciatic nerve, and then sacrificed two weeks later. ( A–D) show sagittal sections of spinal cord with GFAP (blue) outlining the site of spinal transection (arrowhead). Ascending Dextran-labeled sensory axons (arrow) are green. Compared to controls (A), individual treatment with VP16-KLF7 (B) and Lenti-Chase (C) resulted in axon growth that approached nearer to the lesion center. Combined VP16KLF7 and Lenti Chase (D) appeared similar to the individual treatments. E. Quantification of the minimum distance between the lesion center and the nearest axon showed a significant reduction from control in both VP16KLF7 and Lenti-Chase treatments, without significant additive effects. N≥6 animals per group, **p

    Journal: Neurobiology of disease

    Article Title: Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury

    doi: 10.1016/j.nbd.2016.12.010

    Figure Lengend Snippet: VP16-KLF7 and Lenti-chondroitinase increase the proximity of injured sensory axons to sites of spinal injury but do not promote regeneration beyond the injury Sensory neurons in adult mice were transduced by lumbar puncture delivery of AAV-EBFP-2A-mCherry or AAV-VP16-2A-mCherry by lumbar puncture, and received cervical dorsal hemisections accompanied by either control injections or spinal injections of Lenti-Chase. Four weeks later ascending sensory axons were labeled by injection of Dextran-Alexafluor 488 injections to the sciatic nerve, and then sacrificed two weeks later. ( A–D) show sagittal sections of spinal cord with GFAP (blue) outlining the site of spinal transection (arrowhead). Ascending Dextran-labeled sensory axons (arrow) are green. Compared to controls (A), individual treatment with VP16-KLF7 (B) and Lenti-Chase (C) resulted in axon growth that approached nearer to the lesion center. Combined VP16KLF7 and Lenti Chase (D) appeared similar to the individual treatments. E. Quantification of the minimum distance between the lesion center and the nearest axon showed a significant reduction from control in both VP16KLF7 and Lenti-Chase treatments, without significant additive effects. N≥6 animals per group, **p

    Article Snippet: To generate a standard curve of chondroitinase activity, 0.5μg of CSPGs (Millipore) were incubated with chondroitinase enzyme (Amsbio) at 0, 12.5, 25, 50, and 100mU/ml for 2 hours at 37°C, exposed briefly to DMMB, and then 525nM absorbance as quantified by microplate reader (Molecular Devices).

    Techniques: Mouse Assay, Labeling, Injection

    Lenti-Chondroitinase promotes cross-midline sprouting of spared cervical CST axons after unilateral pyramidotomy Unilateral pyramidotomy was performed to deprive the right spinal cord of CST input, and intact CST axons in the left spinal cord were labeled by cortical injecton of AAV8-EGFP. C3/4 spinal cord received injections of PBS control or Lenti-Chase ( A,B) Horizontal sections of cervical spinal cord with labeled CST axons (green) and the midline indicated by the dotted line. Cross-midline sprouting is present in Lenti-chondroitinase treated tissue ( C ) 4 weeks post-injury, significantly more CST axons sprout across the midline in Lenti-chase treated animals. ( D–F ) 2B6 immunohistochemistry shows significant elevation in Lenti-chase treated animals, confirming CSPG degradation. ( G, H ) Show detailed views of sprouting CST axons (green) and 2B6 label (red) in control ( G ) and Lenti-Chase ( H ) tissue. ( I ) Transverse sections of the dorsal columns in thoracic spinal cord were stained with PKC gamma to confirm complete pyramidotomy. N≥4 animals per group, **p

    Journal: Neurobiology of disease

    Article Title: Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury

    doi: 10.1016/j.nbd.2016.12.010

    Figure Lengend Snippet: Lenti-Chondroitinase promotes cross-midline sprouting of spared cervical CST axons after unilateral pyramidotomy Unilateral pyramidotomy was performed to deprive the right spinal cord of CST input, and intact CST axons in the left spinal cord were labeled by cortical injecton of AAV8-EGFP. C3/4 spinal cord received injections of PBS control or Lenti-Chase ( A,B) Horizontal sections of cervical spinal cord with labeled CST axons (green) and the midline indicated by the dotted line. Cross-midline sprouting is present in Lenti-chondroitinase treated tissue ( C ) 4 weeks post-injury, significantly more CST axons sprout across the midline in Lenti-chase treated animals. ( D–F ) 2B6 immunohistochemistry shows significant elevation in Lenti-chase treated animals, confirming CSPG degradation. ( G, H ) Show detailed views of sprouting CST axons (green) and 2B6 label (red) in control ( G ) and Lenti-Chase ( H ) tissue. ( I ) Transverse sections of the dorsal columns in thoracic spinal cord were stained with PKC gamma to confirm complete pyramidotomy. N≥4 animals per group, **p

    Article Snippet: To generate a standard curve of chondroitinase activity, 0.5μg of CSPGs (Millipore) were incubated with chondroitinase enzyme (Amsbio) at 0, 12.5, 25, 50, and 100mU/ml for 2 hours at 37°C, exposed briefly to DMMB, and then 525nM absorbance as quantified by microplate reader (Molecular Devices).

    Techniques: Labeling, Immunohistochemistry, Staining

    Lentiviral transduction drives the secretion of functional chondroitinase in mammalian cells (A) shows the design of plasmid DNA used for the production of Lenti-Chase, indicating mutations to enhance thermal stability and to block aberrant glycosylation. (B) 293T cells were transfected with plasmid encoding EBFP control or chondroitinase, or transduced with Lenti-chase, and the conditioned media tested for chondroitinase activity by colorimetric DMMB assay. Compared to a standard curve of chondroitinase activity (grey circles), media from EBFP-transfected cells showed

    Journal: Neurobiology of disease

    Article Title: Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury

    doi: 10.1016/j.nbd.2016.12.010

    Figure Lengend Snippet: Lentiviral transduction drives the secretion of functional chondroitinase in mammalian cells (A) shows the design of plasmid DNA used for the production of Lenti-Chase, indicating mutations to enhance thermal stability and to block aberrant glycosylation. (B) 293T cells were transfected with plasmid encoding EBFP control or chondroitinase, or transduced with Lenti-chase, and the conditioned media tested for chondroitinase activity by colorimetric DMMB assay. Compared to a standard curve of chondroitinase activity (grey circles), media from EBFP-transfected cells showed

    Article Snippet: To generate a standard curve of chondroitinase activity, 0.5μg of CSPGs (Millipore) were incubated with chondroitinase enzyme (Amsbio) at 0, 12.5, 25, 50, and 100mU/ml for 2 hours at 37°C, exposed briefly to DMMB, and then 525nM absorbance as quantified by microplate reader (Molecular Devices).

    Techniques: Transduction, Functional Assay, Plasmid Preparation, Blocking Assay, Transfection, Activity Assay, Dimethylmethylene Blue Assay

    Chondroitinase secreted by transfected cells reduces inhibition of axon growth by CSPGs (A–D) DRG neurons were cultured for 24 hours on substrates of laminin alone or laminin mixed with CSPG, and treated with conditioned media from 293T cells transfected with control mCherry or Lenti-chondroitinase. CSPG substrate strongly reduced neurite outgrowth ( C ), an effect partially blocked by Lenti-Chase media ( D ). ( E ) shows 48h quantification of DRG neurite growth in 25U/ml chondroitinase enzyme, or in in media conditioned by 293 cells transfected with mCherry plasmid, chondroitinase plasmid, or Lenti-Chase. In control mCherry-transfected conditions, average neurite lengths were reduced by more than 70% at CSPG concentrations of 2μg or higher (p

    Journal: Neurobiology of disease

    Article Title: Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury

    doi: 10.1016/j.nbd.2016.12.010

    Figure Lengend Snippet: Chondroitinase secreted by transfected cells reduces inhibition of axon growth by CSPGs (A–D) DRG neurons were cultured for 24 hours on substrates of laminin alone or laminin mixed with CSPG, and treated with conditioned media from 293T cells transfected with control mCherry or Lenti-chondroitinase. CSPG substrate strongly reduced neurite outgrowth ( C ), an effect partially blocked by Lenti-Chase media ( D ). ( E ) shows 48h quantification of DRG neurite growth in 25U/ml chondroitinase enzyme, or in in media conditioned by 293 cells transfected with mCherry plasmid, chondroitinase plasmid, or Lenti-Chase. In control mCherry-transfected conditions, average neurite lengths were reduced by more than 70% at CSPG concentrations of 2μg or higher (p

    Article Snippet: To generate a standard curve of chondroitinase activity, 0.5μg of CSPGs (Millipore) were incubated with chondroitinase enzyme (Amsbio) at 0, 12.5, 25, 50, and 100mU/ml for 2 hours at 37°C, exposed briefly to DMMB, and then 525nM absorbance as quantified by microplate reader (Molecular Devices).

    Techniques: Transfection, Inhibition, Cell Culture, Plasmid Preparation

    VP16-KLF7 overexpression in CST neurons enhances proximity of the CST to crush injuries independent of chondroitinase treatment A. Cortical neurons in adult mice were transduced by injection of AAV-EBFP-2A-mCherry or AAV-VP16-2A-mCherry, along with AAV-EGFP tracer, and received thoracic crush injury accompanied by either control injections or spinal injections of Lenti-Chase. C–F show sagittal sections of spinal cord with GFAP (blue) showing the injury sites, 2B6 (Red) indicating areas of CSPG degradation, and transduced CST axons (Green, EGFP). Compared to EBFP control (C), VP16-KLF7 transduced axons extend closer to the injury site, but do not extend through the injury center in either the absence (D) or presence (F) of Lenti-chondroitinase. Quantification of the distance between EGFP+ axon tips and the center of the injury shows significant reduction in VP16-KLF7 treated animals. N=9 animals per group, **p

    Journal: Neurobiology of disease

    Article Title: Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury

    doi: 10.1016/j.nbd.2016.12.010

    Figure Lengend Snippet: VP16-KLF7 overexpression in CST neurons enhances proximity of the CST to crush injuries independent of chondroitinase treatment A. Cortical neurons in adult mice were transduced by injection of AAV-EBFP-2A-mCherry or AAV-VP16-2A-mCherry, along with AAV-EGFP tracer, and received thoracic crush injury accompanied by either control injections or spinal injections of Lenti-Chase. C–F show sagittal sections of spinal cord with GFAP (blue) showing the injury sites, 2B6 (Red) indicating areas of CSPG degradation, and transduced CST axons (Green, EGFP). Compared to EBFP control (C), VP16-KLF7 transduced axons extend closer to the injury site, but do not extend through the injury center in either the absence (D) or presence (F) of Lenti-chondroitinase. Quantification of the distance between EGFP+ axon tips and the center of the injury shows significant reduction in VP16-KLF7 treated animals. N=9 animals per group, **p

    Article Snippet: To generate a standard curve of chondroitinase activity, 0.5μg of CSPGs (Millipore) were incubated with chondroitinase enzyme (Amsbio) at 0, 12.5, 25, 50, and 100mU/ml for 2 hours at 37°C, exposed briefly to DMMB, and then 525nM absorbance as quantified by microplate reader (Molecular Devices).

    Techniques: Over Expression, Mouse Assay, Injection

    Effects of increased NG2 expression on neurite growth. A–D , Comparison of the level of NG2 expression in control NG2 cells and in NG2 cells infected with adeno-NG2. A , B , Surface immunofluorescence labeling (live-cell labeling) for NG2 on uninfected ( A ) and adeno-NG2-infected cells ( B ; moi 5). Images were acquired under identical exposure conditions below pixel saturation. Scale bar: (in B ) A , B , 12 μm. C , Quantification of fluorescence intensity of uninfected NG2 cells and NG2 cells infected with adeno-NG2. Cells transduced with adeno-NG2 expressed fivefold more surface NG2 than uninfected NG2 cells. D , Western blots comparing the amount of NG2 in extracts of uninfected and adeno-NG2-infected NG2 cells. The same blot was probed with antibodies to NG2 (top) and GAPDH (bottom) chABC+: extracts treated with chondroitinase ABC. Smaller amounts of protein extracts from adeno-NG2-infected cells were loaded to allow for densitometric analyses of the samples within the dynamic ranges of the pixels, as seen by the weaker GAPDH bands in lanes 3 and 4. The positions of molecular weight standards are indicated on the left. E–I , Response of P1 hippocampal neurons to NG2 cells expressing elevated levels of NG2. E , F , Double-immunofluorescence labeling for NG2 (red) and βIII tubulin (green) to illustrate the spatial relationship between neurites and NG2 cells expressing normal ( E ) or increased levels of surface NG2 after adeno-NG2 transduction ( F ; moi 5). Scale bar: (in F ) E , F , 24 μm. G , Percentage of neurite tips contacting uninfected or adeno-NG2-infected NG2 cells. ns, No statistically significant difference between the number of neurite tips contacting uninfected and adeno-NG2-infected NG2 cells ( P = 0.239, t test). H , Total neurite lengths of neurons grown on uninfected or adeno-NG2-infected NG2 cells. ns, No significant difference ( P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL ( P

    Journal: The Journal of Neuroscience

    Article Title: NG2 Glial Cells Provide a Favorable Substrate for Growing Axons

    doi: 10.1523/JNEUROSCI.4247-05.2006

    Figure Lengend Snippet: Effects of increased NG2 expression on neurite growth. A–D , Comparison of the level of NG2 expression in control NG2 cells and in NG2 cells infected with adeno-NG2. A , B , Surface immunofluorescence labeling (live-cell labeling) for NG2 on uninfected ( A ) and adeno-NG2-infected cells ( B ; moi 5). Images were acquired under identical exposure conditions below pixel saturation. Scale bar: (in B ) A , B , 12 μm. C , Quantification of fluorescence intensity of uninfected NG2 cells and NG2 cells infected with adeno-NG2. Cells transduced with adeno-NG2 expressed fivefold more surface NG2 than uninfected NG2 cells. D , Western blots comparing the amount of NG2 in extracts of uninfected and adeno-NG2-infected NG2 cells. The same blot was probed with antibodies to NG2 (top) and GAPDH (bottom) chABC+: extracts treated with chondroitinase ABC. Smaller amounts of protein extracts from adeno-NG2-infected cells were loaded to allow for densitometric analyses of the samples within the dynamic ranges of the pixels, as seen by the weaker GAPDH bands in lanes 3 and 4. The positions of molecular weight standards are indicated on the left. E–I , Response of P1 hippocampal neurons to NG2 cells expressing elevated levels of NG2. E , F , Double-immunofluorescence labeling for NG2 (red) and βIII tubulin (green) to illustrate the spatial relationship between neurites and NG2 cells expressing normal ( E ) or increased levels of surface NG2 after adeno-NG2 transduction ( F ; moi 5). Scale bar: (in F ) E , F , 24 μm. G , Percentage of neurite tips contacting uninfected or adeno-NG2-infected NG2 cells. ns, No statistically significant difference between the number of neurite tips contacting uninfected and adeno-NG2-infected NG2 cells ( P = 0.239, t test). H , Total neurite lengths of neurons grown on uninfected or adeno-NG2-infected NG2 cells. ns, No significant difference ( P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL ( P

    Article Snippet: Some extracts were treated with 0.125 U of chondroitinase ABC (MP Biomedicals, Aurora, OH) at room temperature for an additional 15 min. Extracts were then centrifuged at 15,000 × g for 5 min at 4°C to remove insoluble material, and supernatants were heated at 80°C for 10 min in reducing sample buffer (6% SDS, 40% glycerol, and 0.2 mg/ml BPB, and 100 m m DTT in 125 m m Tris-HCl, pH 6.8).

    Techniques: Expressing, Infection, Immunofluorescence, Labeling, Fluorescence, Transduction, Western Blot, Molecular Weight