flavobacterium heparinum Search Results


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  • 99
    Millipore heparinase i
    LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither <t>heparinase</t> ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.
    Heparinase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore heparinase iii
    Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without <t>heparinase</t> <t>III</t> for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p
    Heparinase Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 776 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore flavobacterium heparinum
    Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase <t>I/III</t> blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P
    Flavobacterium Heparinum, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore heparinase ii
    Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase <t>I/III</t> blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P
    Heparinase Ii, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore chondroitinase
    Treatment of RD cells with heparinase or <t>chondroitinase</t> <t>ABC.</t> (A) Inhibitory effects of heparinase I/II/III and chondroitinase ABC on EV-71 infection. RD cells were pretreated with heparinase or chondroitinase ABC for 1 h at 37°C before EV-71 infection
    Chondroitinase, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Seikagaku flavobacterium heparinum
    Treatment of RD cells with heparinase or <t>chondroitinase</t> <t>ABC.</t> (A) Inhibitory effects of heparinase I/II/III and chondroitinase ABC on EV-71 infection. RD cells were pretreated with heparinase or chondroitinase ABC for 1 h at 37°C before EV-71 infection
    Flavobacterium Heparinum, supplied by Seikagaku, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore chondroitinase b
    Treatment of RD cells with heparinase or <t>chondroitinase</t> <t>ABC.</t> (A) Inhibitory effects of heparinase I/II/III and chondroitinase ABC on EV-71 infection. RD cells were pretreated with heparinase or chondroitinase ABC for 1 h at 37°C before EV-71 infection
    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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    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 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    IBEX Technologies flavobacterium heparinum
    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.
    Flavobacterium Heparinum, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither heparinase ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.

    Journal: Glycobiology

    Article Title: LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans

    doi: 10.1093/glycob/cww075

    Figure Lengend Snippet: LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither heparinase ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.

    Article Snippet: Heparinase treatment and HF dephosphorylation For heparinase treatment, samples were treated with a mixture of heparinases I and III (Sigma, 0.6 and 0.3 Sigma units, respectively) in 0.

    Techniques: Binding Assay, Construct, Sequencing, Purification, Expressing, Functional Assay, Modification

    Characterization of SDC 4–promyostatin interaction. (A) Co‐immunoprecipitations (co‐ IP s) were carried out with rabbit antiserum to the C‐terminal part of myostatin ( AB 3239) in un‐injured (control, day 0, d0) and injured (3 days after notexin injury, d3) soleus muscle homogenates. Different volumes (20 and 7 μL) of the eluted immunocomplex were loaded in case of the injured sample. The blots were reacted with antibodies to SDC 4 raised in goat. Myostatin co‐immunoprecipitated SDC 4 in both cases. Input lanes represent the total homogenates; 10% of the total protein amount used in co‐ IP was loaded. (B) Co‐ IP assays were performed with anti‐ SDC 4 antibodies, and the blots were reacted with anti‐myostatin antisera ( AB 3239). The negative control was incubated only with the secondary antibody. For the input lanes, 10% of the total protein amount used in co‐ IP was loaded. The additional band at ~ 42 kD a in d0 input can be a processing intermediate of promyostatin. (C) Heparan sulfate chains were digested in injured samples (d3; sample 1 and 2) with heparinase II enzyme following immunoprecipitation with goat anti‐ SDC 4 antibody. Different amounts (25 and 5 μL) of the eluted volume of the heparinase II digested immunoprecipitate of sample 1 were loaded. We could not detect promyostatin in the immunoprecipitate after heparinase digestion. For the input lanes, 7.5% of the total protein amount used in co‐ IP s were loaded. Note, that both promyostatin and mature myostatin were detected in the supernatant (digestion buffer) after heparinase digestion.

    Journal: Febs Letters

    Article Title: Syndecan‐4 influences mammalian myoblast proliferation by modulating myostatin signalling and G1/S transition

    doi: 10.1002/1873-3468.13227

    Figure Lengend Snippet: Characterization of SDC 4–promyostatin interaction. (A) Co‐immunoprecipitations (co‐ IP s) were carried out with rabbit antiserum to the C‐terminal part of myostatin ( AB 3239) in un‐injured (control, day 0, d0) and injured (3 days after notexin injury, d3) soleus muscle homogenates. Different volumes (20 and 7 μL) of the eluted immunocomplex were loaded in case of the injured sample. The blots were reacted with antibodies to SDC 4 raised in goat. Myostatin co‐immunoprecipitated SDC 4 in both cases. Input lanes represent the total homogenates; 10% of the total protein amount used in co‐ IP was loaded. (B) Co‐ IP assays were performed with anti‐ SDC 4 antibodies, and the blots were reacted with anti‐myostatin antisera ( AB 3239). The negative control was incubated only with the secondary antibody. For the input lanes, 10% of the total protein amount used in co‐ IP was loaded. The additional band at ~ 42 kD a in d0 input can be a processing intermediate of promyostatin. (C) Heparan sulfate chains were digested in injured samples (d3; sample 1 and 2) with heparinase II enzyme following immunoprecipitation with goat anti‐ SDC 4 antibody. Different amounts (25 and 5 μL) of the eluted volume of the heparinase II digested immunoprecipitate of sample 1 were loaded. We could not detect promyostatin in the immunoprecipitate after heparinase digestion. For the input lanes, 7.5% of the total protein amount used in co‐ IP s were loaded. Note, that both promyostatin and mature myostatin were detected in the supernatant (digestion buffer) after heparinase digestion.

    Article Snippet: The immunoprecipitate was resuspended in heparinase buffer and digested with 0.4 mU heparinase II enzyme (Sigma‐Aldrich) for 3 h at 37 °C.

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Flow Cytometry, DCFH-DA Assay, Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in SH-SY5Y cell morphology and cell viability. ( a ) SH-SY5Y cell morphology. Bar, 10 μM. Images were analyzed with SPOT 4.7 Advanced software. The arrows indicate the shorter neurite outgrowth of SH-SY5Y cells after the Aβ(25-35) challenge. ( b ) SH-SY5Y cell viability was analyzed by an MTT assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in SH-SY5Y cell morphology and cell viability. ( a ) SH-SY5Y cell morphology. Bar, 10 μM. Images were analyzed with SPOT 4.7 Advanced software. The arrows indicate the shorter neurite outgrowth of SH-SY5Y cells after the Aβ(25-35) challenge. ( b ) SH-SY5Y cell viability was analyzed by an MTT assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Software, MTT Assay, Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in the phosphorylated (p)-tau/tau ratio ( a ), phosphorylated (p)-phosphatidylinositol 3K (PI3K)/PI3K ratio ( b ), the phosphorylated (p)-Akt/Akt ratio ( c ), and the phosphorylated (p)-glycogen synthase kinase (GSK)-3β (Ser 9 )/GSK-3β ratio ( d ) of protein expressions. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in the phosphorylated (p)-tau/tau ratio ( a ), phosphorylated (p)-phosphatidylinositol 3K (PI3K)/PI3K ratio ( b ), the phosphorylated (p)-Akt/Akt ratio ( c ), and the phosphorylated (p)-glycogen synthase kinase (GSK)-3β (Ser 9 )/GSK-3β ratio ( d ) of protein expressions. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in cell apoptosis. ( a ) Western blot analysis of caspase-3 protein expression in SH-SY5Y cells. ( b ) Percentages of apoptotic cells in each group quantified from ( c ). ( c ) Representative profiles of cell apoptosis detected by flow cytometry with Annexin V/propidium iodide double-staining. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in cell apoptosis. ( a ) Western blot analysis of caspase-3 protein expression in SH-SY5Y cells. ( b ) Percentages of apoptotic cells in each group quantified from ( c ). ( c ) Representative profiles of cell apoptosis detected by flow cytometry with Annexin V/propidium iodide double-staining. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Western Blot, Expressing, Flow Cytometry, Double Staining, Incubation

    Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with heparitinase III, as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: IL-1 and TGF-β Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells

    doi: 10.1167/iovs.18-25202

    Figure Lengend Snippet: Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with heparitinase III, as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P

    Article Snippet: For perlecan Western blots, the dialyzed extract was digested with 1 mU/mL of heparitinase III (Cat No. H8891; Sigma-Aldrich) at 37°C for 3 hours.

    Techniques: Expressing, Cell Culture, Western Blot

    Double-labeled ( closed circles , [ 3 H]glucosamine; open circles , [ 35 S]sulfate) β-TC3 HS following application to and elution from an IAPP affinity column, and following heparinase I or heparinase III digestion. Reaction products were separated by Bio-gel P-10 size exclusion chromatography. IAPP-bound HS is shown in A (heparinase I digested) and B (heparinase III digested), whereas non-bound HS is shown in C (heparinase I digested) and D (heparinase III digested). β-TC3 HS showed similar composition regardless of IAPP binding ability, with both bound and non-bound material being relatively insensitive to heparinase I treatment but sensitive to heparinase III treatment.

    Journal: The Journal of Biological Chemistry

    Article Title: Overall Sulfation of Heparan Sulfate from Pancreatic Islet ?-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide *

    doi: 10.1074/jbc.M112.409847

    Figure Lengend Snippet: Double-labeled ( closed circles , [ 3 H]glucosamine; open circles , [ 35 S]sulfate) β-TC3 HS following application to and elution from an IAPP affinity column, and following heparinase I or heparinase III digestion. Reaction products were separated by Bio-gel P-10 size exclusion chromatography. IAPP-bound HS is shown in A (heparinase I digested) and B (heparinase III digested), whereas non-bound HS is shown in C (heparinase I digested) and D (heparinase III digested). β-TC3 HS showed similar composition regardless of IAPP binding ability, with both bound and non-bound material being relatively insensitive to heparinase I treatment but sensitive to heparinase III treatment.

    Article Snippet: Heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycans were selectively removed from the immobilized proteoglycans by heparinase digestion (2.6 units/ml heparinase I, 1.3 units/ml heparinase II + 2.6 units/ml heparinase III, Sigma) or chondroitin ABC lyase digestion (0.1 units/ml), respectively, for 2 h at 37 °C.

    Techniques: Labeling, Affinity Column, Size-exclusion Chromatography, Binding Assay

    Double-labeled ( closed symbols , [ 3 H]glucosamine; open symbols , [ 35 S]sulfate) HS from β-TC3 cells ( A ) and NMuMG cells ( B ) was subjected to Sepharose CL-6B size exclusion chromatography prior to ( circles ) or following heparinase I digestion ( triangles ), which cleaves highly sulfated regions of heparan sulfate. Intact β-TC3 heparan sulfate eluted at K av = 0.6, whereas two HS peaks of approximately equal abundance were observed after heparinase I treatment: at K av = 0.58 and 0.8. Thus, β-TC3 HS was only partially sensitive to heparinase I digestion, demonstrating poor overall sulfation. Intact NMuMG HS eluted at K av = 0.45, whereas following heparinase I digestion, a larger peak was present at K av = 0.7 and a smaller peak at K av = 0.9. Thus, NMuMG HS was more sensitive to heparinase I digestion, demonstrating higher overall sulfation in this HS preparation.

    Journal: The Journal of Biological Chemistry

    Article Title: Overall Sulfation of Heparan Sulfate from Pancreatic Islet ?-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide *

    doi: 10.1074/jbc.M112.409847

    Figure Lengend Snippet: Double-labeled ( closed symbols , [ 3 H]glucosamine; open symbols , [ 35 S]sulfate) HS from β-TC3 cells ( A ) and NMuMG cells ( B ) was subjected to Sepharose CL-6B size exclusion chromatography prior to ( circles ) or following heparinase I digestion ( triangles ), which cleaves highly sulfated regions of heparan sulfate. Intact β-TC3 heparan sulfate eluted at K av = 0.6, whereas two HS peaks of approximately equal abundance were observed after heparinase I treatment: at K av = 0.58 and 0.8. Thus, β-TC3 HS was only partially sensitive to heparinase I digestion, demonstrating poor overall sulfation. Intact NMuMG HS eluted at K av = 0.45, whereas following heparinase I digestion, a larger peak was present at K av = 0.7 and a smaller peak at K av = 0.9. Thus, NMuMG HS was more sensitive to heparinase I digestion, demonstrating higher overall sulfation in this HS preparation.

    Article Snippet: Heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycans were selectively removed from the immobilized proteoglycans by heparinase digestion (2.6 units/ml heparinase I, 1.3 units/ml heparinase II + 2.6 units/ml heparinase III, Sigma) or chondroitin ABC lyase digestion (0.1 units/ml), respectively, for 2 h at 37 °C.

    Techniques: Labeling, Size-exclusion Chromatography

    HS plays a role in henipavirus infection. (A) Vero cells were either treated with heparinase 3 or left untreated prior to NiV infection. Titration was performed 3 days later in a plaque assay. (B and C) Vero cells were treated with increasing concentrations of sodium chlorate for 48 h and then infected with either NiV (B) or HeV (C); titration was performed 3 days later in a plaque assay. Results are expressed as a percentage of results for nontreated controls from triplicate cultures, ± the SD. *, P

    Journal: mBio

    Article Title: Heparan Sulfate-Dependent Enhancement of Henipavirus Infection

    doi: 10.1128/mBio.02427-14

    Figure Lengend Snippet: HS plays a role in henipavirus infection. (A) Vero cells were either treated with heparinase 3 or left untreated prior to NiV infection. Titration was performed 3 days later in a plaque assay. (B and C) Vero cells were treated with increasing concentrations of sodium chlorate for 48 h and then infected with either NiV (B) or HeV (C); titration was performed 3 days later in a plaque assay. Results are expressed as a percentage of results for nontreated controls from triplicate cultures, ± the SD. *, P

    Article Snippet: In some experiments, cells were incubated for 1 h at 37°C with 10 U/ml of heparinase 3 (Sigma) and washed 3 times before contact with NiV.

    Techniques: Infection, Titration, Plaque Assay

    trans -Infection with henipaviruses requires the expression of HS. (A) Lymphocyte-mediated  trans -infection by NiV and HeV. PBLs were incubated with either NiV or HeV, washed, cultured for 24 h, and then transferred to Vero cell monolayers, which were used for the determination of the viral titers after 4 days of coculture, using infectious center assays. (B) CHO-K1 cells, treated or not with heparinase 3, and three HS-deficient CHO lines, pgsA-745, pgsB-619, and pgsD-677, were incubated with NiV and analyzed for their capacities to transmit infection to susceptible Vero cells in  trans . Results are expressed as a percentage of inhibition compared to results with untreated cells ± SD. *,  P

    Journal: mBio

    Article Title: Heparan Sulfate-Dependent Enhancement of Henipavirus Infection

    doi: 10.1128/mBio.02427-14

    Figure Lengend Snippet: trans -Infection with henipaviruses requires the expression of HS. (A) Lymphocyte-mediated trans -infection by NiV and HeV. PBLs were incubated with either NiV or HeV, washed, cultured for 24 h, and then transferred to Vero cell monolayers, which were used for the determination of the viral titers after 4 days of coculture, using infectious center assays. (B) CHO-K1 cells, treated or not with heparinase 3, and three HS-deficient CHO lines, pgsA-745, pgsB-619, and pgsD-677, were incubated with NiV and analyzed for their capacities to transmit infection to susceptible Vero cells in trans . Results are expressed as a percentage of inhibition compared to results with untreated cells ± SD. *, P

    Article Snippet: In some experiments, cells were incubated for 1 h at 37°C with 10 U/ml of heparinase 3 (Sigma) and washed 3 times before contact with NiV.

    Techniques: Infection, Expressing, Incubation, Cell Culture, Inhibition

    Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase I/III blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P

    Journal: Cell Adhesion & Migration

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

    doi: 10.1080/19336918.2015.1103421

    Figure Lengend Snippet: Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase I/III blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P

    Article Snippet: Treatment of cells with heparinase I/III and sodium chlorate To assess the influence of surface heparan sulfate digestion on the cell surface expression of Hsp90α and Hsp90β, A-172 and HT1080 cells were washed with DMEM and incubated for 1 h at 37°C with a heparinase I/III blend from Flavobacterium heparinum (Sigma-Aldrich) diluted in DMEM-FBS (0.03 IU/ml).

    Techniques: Incubation, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Negative Control, Expressing

    GPR158 is a heparan sulfate-dependent GPC4 binding partner (A) Proteomic workflow for the identification of GPC4-interacting proteins. (B) Identification of GPR158 as a GPC4 interactor by tandem mass spectrometry. GPC4-Fc protein was used as bait and synaptosome extract from P21 rat brains as prey. Graph shows summed peptide and spectral counts for all surface proteins after Fc background subtraction (n = 3 independent experiments). (C) Freqency of detection of peptides (spectral count) for all proteins identified in two independent GPC4-Fc affinity purification experiments after Fc background subtraction. (D) GPR158 domain organization. LRD, Leucine-Rich Domain; EGF-like, EGF-like Ca 2+ binding motif. Yellow region, RGS7-binding site. (E) Cell-surface binding assays in HEK293T cells. GPC4-Fc (red), but not GPC4 ΔGAG-Fc, binds the GPR158 ectodomain (green) expressed on the cell membrane. LRRTM4 and EGFP serve as positive and negative controls, respectively. (F) Pulldown assay in HEK293T cells. GPC4-Fc, but not Fc alone, binds GPR158. HS removal by mutagenesis (GPC4 ΔGAG-Fc) or Heparinase III treatment (GPC4-Fc/HepIII) abolishes the interaction. (G) GPC4 and GPR158 interact in trans . GPC4 co-immunoprecipitates with GPR158 following expression in separately transfected and co-cultured HEK293T cells. 65 kDa band represents full-length GPC4; 40 kDa band the N-terminal proteolytic fragment. IgG serves as negative control. (H) Pulldown assay in hippocampal lysate. GPR158-Fc, but not Fc alone, binds endogenous GPC4. Scale bar in (E) 10 µm. .

    Journal: Neuron

    Article Title: An input-specific orphan receptor GPR158-HSPG interaction organizes hippocampal mossy fiber-CA3 synapses

    doi: 10.1016/j.neuron.2018.08.038

    Figure Lengend Snippet: GPR158 is a heparan sulfate-dependent GPC4 binding partner (A) Proteomic workflow for the identification of GPC4-interacting proteins. (B) Identification of GPR158 as a GPC4 interactor by tandem mass spectrometry. GPC4-Fc protein was used as bait and synaptosome extract from P21 rat brains as prey. Graph shows summed peptide and spectral counts for all surface proteins after Fc background subtraction (n = 3 independent experiments). (C) Freqency of detection of peptides (spectral count) for all proteins identified in two independent GPC4-Fc affinity purification experiments after Fc background subtraction. (D) GPR158 domain organization. LRD, Leucine-Rich Domain; EGF-like, EGF-like Ca 2+ binding motif. Yellow region, RGS7-binding site. (E) Cell-surface binding assays in HEK293T cells. GPC4-Fc (red), but not GPC4 ΔGAG-Fc, binds the GPR158 ectodomain (green) expressed on the cell membrane. LRRTM4 and EGFP serve as positive and negative controls, respectively. (F) Pulldown assay in HEK293T cells. GPC4-Fc, but not Fc alone, binds GPR158. HS removal by mutagenesis (GPC4 ΔGAG-Fc) or Heparinase III treatment (GPC4-Fc/HepIII) abolishes the interaction. (G) GPC4 and GPR158 interact in trans . GPC4 co-immunoprecipitates with GPR158 following expression in separately transfected and co-cultured HEK293T cells. 65 kDa band represents full-length GPC4; 40 kDa band the N-terminal proteolytic fragment. IgG serves as negative control. (H) Pulldown assay in hippocampal lysate. GPR158-Fc, but not Fc alone, binds endogenous GPC4. Scale bar in (E) 10 µm. .

    Article Snippet: For the analysis of heparinase III treatment, hippocampal neurons (7 DIV) were treated with 1 U/ml heparinase III (Sigma-Aldrich) or vehicle (20 mM Tris-HCl [pH 7.5], 0.1 mg/ml BSA, 4mM CaCl2 ) for 2 hours at 37°C.

    Techniques: Binding Assay, Mass Spectrometry, Affinity Purification, Mutagenesis, Expressing, Transfection, Cell Culture, Negative Control

    Treatment of RD cells with heparinase or chondroitinase ABC. (A) Inhibitory effects of heparinase I/II/III and chondroitinase ABC on EV-71 infection. RD cells were pretreated with heparinase or chondroitinase ABC for 1 h at 37°C before EV-71 infection

    Journal: Journal of Virology

    Article Title: Enterovirus 71 Uses Cell Surface Heparan Sulfate Glycosaminoglycan as an Attachment Receptor

    doi: 10.1128/JVI.02226-12

    Figure Lengend Snippet: Treatment of RD cells with heparinase or chondroitinase ABC. (A) Inhibitory effects of heparinase I/II/III and chondroitinase ABC on EV-71 infection. RD cells were pretreated with heparinase or chondroitinase ABC for 1 h at 37°C before EV-71 infection

    Article Snippet: Soluble GAGs (heparin sodium salt from porcine intestinal mucosa, chondroitin sulfate sodium salt from shark cartilage, N -acetyl-de-O-sulfated heparin sodium salt, and de-N-sulfated heparin sodium salt), dextran sulfate sodium salt from Leuconostoc mesenteroides , suramin, heparinase I/II/III from Flavobacterium heparinum , chondroitinase ABC from Proteus vulgaris , and sodium chlorate were all purchased from Sigma.

    Techniques: Infection

    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