Structured Review

Millipore pustulan
β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of <t>pustulan</t> (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P
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1) Product Images from "A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi"

Article Title: A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121801

β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P
Figure Legend Snippet: β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P

Techniques Used: Staining, Expressing

Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.
Figure Legend Snippet: Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.

Techniques Used: Recombinant, Binding Assay, Dot Blot, Staining

V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. k a in 10 4 sec −1 M −1 , k d in 10 −5 sec −1 , K D in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants ( k a ) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.
Figure Legend Snippet: V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. k a in 10 4 sec −1 M −1 , k d in 10 −5 sec −1 , K D in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants ( k a ) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.

Techniques Used: SPR Assay, Binding Assay, Size-exclusion Chromatography, Molecular Weight

2) Product Images from "Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans"

Article Title: Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans

Journal: Molecular Microbiology

doi: 10.1111/j.1365-2958.2010.07210.x

Hst 5 binds selectively to β-1,3-glucans in the Candida cell wall. Representative cell wall polysaccharides (1–16 mg ml −1 ) consisting of laminarin (β-1,3-glucan) (Lam, red squares), sialic acid (Sia, grey circles), mannans (Man, black diamonds), pustulan (β-1,6-glucan) (Pus, black squares) or untreated control (Con) were pre-incubated with BHst 5 (31 µM) then added to C. albicans cells for 1 h. Cell wall extracts were immunoblotted to detect BHst 5 (A) and quantified by dentistometry (B). Reduction in cell wall binding was accompanied by reduced candidacidal activity of Hst 5 when incubated with the same polysaccharides in a dose-dependent manner (C). The same polysaccharides were coupled to Sepharose beads and percent binding of BHst 5 was assessed by elution affinity chromatography (D). Only laminarin was found to have significant binding with Hst 5. PCW and laminarinase-treated PCW were incubated with Hst 5, and unbound Hst 5 in supernatant (S) and bound Hst 5 in pellets (P) were detected on Tricine-SDS gels (E). Laminarinase digestion of PCW prevented Hst 5 binding (E). Cell wall binding of Hst 5 was reduced in a dose-dependent manner by laminarinase digests (F), when laminarinase digests (2–8 mg ml −1 ) were pre-incubated with Hst 5, then added to C. albicans cells. Killing activities of Hst 5 were decreased against C. albicans cells grown in YPD broth with caspofungin (1 or 2 ng ml −1 ) (G).
Figure Legend Snippet: Hst 5 binds selectively to β-1,3-glucans in the Candida cell wall. Representative cell wall polysaccharides (1–16 mg ml −1 ) consisting of laminarin (β-1,3-glucan) (Lam, red squares), sialic acid (Sia, grey circles), mannans (Man, black diamonds), pustulan (β-1,6-glucan) (Pus, black squares) or untreated control (Con) were pre-incubated with BHst 5 (31 µM) then added to C. albicans cells for 1 h. Cell wall extracts were immunoblotted to detect BHst 5 (A) and quantified by dentistometry (B). Reduction in cell wall binding was accompanied by reduced candidacidal activity of Hst 5 when incubated with the same polysaccharides in a dose-dependent manner (C). The same polysaccharides were coupled to Sepharose beads and percent binding of BHst 5 was assessed by elution affinity chromatography (D). Only laminarin was found to have significant binding with Hst 5. PCW and laminarinase-treated PCW were incubated with Hst 5, and unbound Hst 5 in supernatant (S) and bound Hst 5 in pellets (P) were detected on Tricine-SDS gels (E). Laminarinase digestion of PCW prevented Hst 5 binding (E). Cell wall binding of Hst 5 was reduced in a dose-dependent manner by laminarinase digests (F), when laminarinase digests (2–8 mg ml −1 ) were pre-incubated with Hst 5, then added to C. albicans cells. Killing activities of Hst 5 were decreased against C. albicans cells grown in YPD broth with caspofungin (1 or 2 ng ml −1 ) (G).

Techniques Used: Laser Capture Microdissection, Incubation, Binding Assay, Activity Assay, Affinity Chromatography

3) Product Images from "Interactions of Surfactant Proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus"

Article Title: Interactions of Surfactant Proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus

Journal: Infection and Immunity

doi: 10.1128/IAI.69.4.2037-2044.2001

Glucosyl polysaccharides laminarin and pustulan. The carbon atom numbering schemes for each are indicated.
Figure Legend Snippet: Glucosyl polysaccharides laminarin and pustulan. The carbon atom numbering schemes for each are indicated.

Techniques Used:

Inhibition of SP-D-induced yeast aggregation. Yeast cells were suspended in calcium-containing buffer with or without carbohydrate inhibitor at room temperature. After 5 min, recombinant human SP-D was added to all samples except the negative control (final SP-D concentration was 5 μg/ml). Buffer was added to the negative control sample. The A 700 of the suspensions was monitored every minute for 2 h after protein addition. For the graphs shown, the starting A 700 for all samples was normalized to the buffer control for ease of interpretation. Maltose, pustulan, and laminarin concentrations are reported as glucose equivalents (Glc eq.). The graph shows representative data from duplicate experiments.
Figure Legend Snippet: Inhibition of SP-D-induced yeast aggregation. Yeast cells were suspended in calcium-containing buffer with or without carbohydrate inhibitor at room temperature. After 5 min, recombinant human SP-D was added to all samples except the negative control (final SP-D concentration was 5 μg/ml). Buffer was added to the negative control sample. The A 700 of the suspensions was monitored every minute for 2 h after protein addition. For the graphs shown, the starting A 700 for all samples was normalized to the buffer control for ease of interpretation. Maltose, pustulan, and laminarin concentrations are reported as glucose equivalents (Glc eq.). The graph shows representative data from duplicate experiments.

Techniques Used: Inhibition, Recombinant, Negative Control, Concentration Assay, Gas Chromatography

4) Product Images from "Endophytic Fungal ?-1,6-Glucanase Expression in the Infected Host Grass 1"

Article Title: Endophytic Fungal ?-1,6-Glucanase Expression in the Infected Host Grass 1

Journal: Plant Physiology

doi: 10.1104/pp.010108

RNA gel-blot analysis of β-1,6-glucanase transcripts. A, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, Neotyphodium sp. grown on 0.5% (w/v) pustulan; lane 4, Neotyphodium sp. grown on 0.5% (w/v) laminarin. Fifteen micrograms of total RNA was used from E− and E+ P. ampla leaf sheaths. Ten micrograms of total RNA was used from the Neotyphodium sp. fungal cultures. B, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, E+ Chewings fescue; lane 4, E+ tall fescue; lane 5, Chewings fescue 1117 artificially infected with the Neotyphodium sp. Four micrograms of poly(A + ) RNA was used for each sample.
Figure Legend Snippet: RNA gel-blot analysis of β-1,6-glucanase transcripts. A, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, Neotyphodium sp. grown on 0.5% (w/v) pustulan; lane 4, Neotyphodium sp. grown on 0.5% (w/v) laminarin. Fifteen micrograms of total RNA was used from E− and E+ P. ampla leaf sheaths. Ten micrograms of total RNA was used from the Neotyphodium sp. fungal cultures. B, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, E+ Chewings fescue; lane 4, E+ tall fescue; lane 5, Chewings fescue 1117 artificially infected with the Neotyphodium sp. Four micrograms of poly(A + ) RNA was used for each sample.

Techniques Used: Western Blot, Infection

5) Product Images from "Endophytic Fungal ?-1,6-Glucanase Expression in the Infected Host Grass 1"

Article Title: Endophytic Fungal ?-1,6-Glucanase Expression in the Infected Host Grass 1

Journal: Plant Physiology

doi: 10.1104/pp.010108

RNA gel-blot analysis of β-1,6-glucanase transcripts. A, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, Neotyphodium sp. grown on 0.5% (w/v) pustulan; lane 4, Neotyphodium sp. grown on 0.5% (w/v) laminarin. Fifteen micrograms of total RNA was used from E− and E+ P. ampla leaf sheaths. Ten micrograms of total RNA was used from the Neotyphodium sp. fungal cultures. B, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, E+ Chewings fescue; lane 4, E+ tall fescue; lane 5, Chewings fescue 1117 artificially infected with the Neotyphodium sp. Four micrograms of poly(A + ) RNA was used for each sample.
Figure Legend Snippet: RNA gel-blot analysis of β-1,6-glucanase transcripts. A, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, Neotyphodium sp. grown on 0.5% (w/v) pustulan; lane 4, Neotyphodium sp. grown on 0.5% (w/v) laminarin. Fifteen micrograms of total RNA was used from E− and E+ P. ampla leaf sheaths. Ten micrograms of total RNA was used from the Neotyphodium sp. fungal cultures. B, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, E+ Chewings fescue; lane 4, E+ tall fescue; lane 5, Chewings fescue 1117 artificially infected with the Neotyphodium sp. Four micrograms of poly(A + ) RNA was used for each sample.

Techniques Used: Western Blot, Infection

6) Product Images from "The N-terminal Domain of Drosophila Gram-negative Binding Protein 3 (GNBP3) Defines a Novel Family of Fungal Pattern Recognition Receptors *"

Article Title: The N-terminal Domain of Drosophila Gram-negative Binding Protein 3 (GNBP3) Defines a Novel Family of Fungal Pattern Recognition Receptors *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.034587

Binding of GNBP3-Nter to polysaccharides versus oligosaccharides. A , direct ELISA assays showing the binding of GNPB3-Nter to the cell wall AI fractions of S. cerevisiae ( ScAI ) and A. fumigatus ( AfAI ), AS fraction of A. fumigatus ( AfAS ), and other commercially available fungal cell wall polysaccharides. Note that GNBP3-Nter does not bind to chitin, pustulan, or the AS fraction of A. fumigatus cell wall. Binding with schizophyllan (a highly branched β-glucan) is ∼1/10 of curdlan, confirming the affinity of GNBP3-Nter for linear β-(1,3)-glucan. Each bar represents mean ± S.D. of six repetitions. B , ELISA inhibition assays show that linear oligosaccharides of DP > 20 are the best inhibitor for binding of GNBP3-Nter to AI fractions of A. fumigatus at 1–400 μg/0.5 μg of GNBP3-Nter. Each bar represents the mean ± S.D. of four repetitions, black *, p
Figure Legend Snippet: Binding of GNBP3-Nter to polysaccharides versus oligosaccharides. A , direct ELISA assays showing the binding of GNPB3-Nter to the cell wall AI fractions of S. cerevisiae ( ScAI ) and A. fumigatus ( AfAI ), AS fraction of A. fumigatus ( AfAS ), and other commercially available fungal cell wall polysaccharides. Note that GNBP3-Nter does not bind to chitin, pustulan, or the AS fraction of A. fumigatus cell wall. Binding with schizophyllan (a highly branched β-glucan) is ∼1/10 of curdlan, confirming the affinity of GNBP3-Nter for linear β-(1,3)-glucan. Each bar represents mean ± S.D. of six repetitions. B , ELISA inhibition assays show that linear oligosaccharides of DP > 20 are the best inhibitor for binding of GNBP3-Nter to AI fractions of A. fumigatus at 1–400 μg/0.5 μg of GNBP3-Nter. Each bar represents the mean ± S.D. of four repetitions, black *, p

Techniques Used: Binding Assay, Direct ELISA, Enzyme-linked Immunosorbent Assay, Inhibition

7) Product Images from "Analysis of the ?-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum"

Article Title: Analysis of the ?-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

Journal: Applied and Environmental Microbiology

doi:

Effect of carbon source on the production of β-1,3-glucanase by T. harzianum . Culture filtrates were obtained by using mineral medium supplemented with cell walls from M. rouxii (bar 1), N. crassa (bar 2), R. solani (bar 3), or S. cerevisiae (bar 4), pustulan (bar 5), pullulan (bar 6), laminarin (bar 7), filtrate of autoclaved S. cerevisiae cell walls (bar 8), or glucose (bar 9). β-1,3-Glucanase activity was determined as described in the text.
Figure Legend Snippet: Effect of carbon source on the production of β-1,3-glucanase by T. harzianum . Culture filtrates were obtained by using mineral medium supplemented with cell walls from M. rouxii (bar 1), N. crassa (bar 2), R. solani (bar 3), or S. cerevisiae (bar 4), pustulan (bar 5), pullulan (bar 6), laminarin (bar 7), filtrate of autoclaved S. cerevisiae cell walls (bar 8), or glucose (bar 9). β-1,3-Glucanase activity was determined as described in the text.

Techniques Used: Activity Assay

IEF of β-1,3-glucanase from T. harzianum filtrates. Culture filtrates were obtained by using mineral medium supplemented with different commercial polysaccharides or fungal cell walls as sole carbon sources. (A) Culture filtrates precipitated with acetone. Lane 1, glucose; lane 2, M. rouxii cell walls; lane 3, N. crassa cell walls; lane 4, R. solani cell walls; lane 5, S. cerevisiae cell walls; lane 6, S. cerevisiae cell wall filtrate; lane 7, S. cerevisiae residual cell walls; lane 8, pustulan; lane 9, laminarin; lane 10, pullulan. Lanes were loaded with 1 U of enzyme. (B) Culture filtrates that were dialyzed and lyophilized. Lane 1, laminarin; lane 2, R. solani ; lane 3, glucose. All lanes were loaded with 15 μg of protein.
Figure Legend Snippet: IEF of β-1,3-glucanase from T. harzianum filtrates. Culture filtrates were obtained by using mineral medium supplemented with different commercial polysaccharides or fungal cell walls as sole carbon sources. (A) Culture filtrates precipitated with acetone. Lane 1, glucose; lane 2, M. rouxii cell walls; lane 3, N. crassa cell walls; lane 4, R. solani cell walls; lane 5, S. cerevisiae cell walls; lane 6, S. cerevisiae cell wall filtrate; lane 7, S. cerevisiae residual cell walls; lane 8, pustulan; lane 9, laminarin; lane 10, pullulan. Lanes were loaded with 1 U of enzyme. (B) Culture filtrates that were dialyzed and lyophilized. Lane 1, laminarin; lane 2, R. solani ; lane 3, glucose. All lanes were loaded with 15 μg of protein.

Techniques Used: Electrofocusing

8) Product Images from "Endophytic Fungal ?-1,6-Glucanase Expression in the Infected Host Grass 1"

Article Title: Endophytic Fungal ?-1,6-Glucanase Expression in the Infected Host Grass 1

Journal: Plant Physiology

doi: 10.1104/pp.010108

RNA gel-blot analysis of β-1,6-glucanase transcripts. A, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, Neotyphodium sp. grown on 0.5% (w/v) pustulan; lane 4, Neotyphodium sp. grown on 0.5% (w/v) laminarin. Fifteen micrograms of total RNA was used from E− and E+ P. ampla leaf sheaths. Ten micrograms of total RNA was used from the Neotyphodium sp. fungal cultures. B, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, E+ Chewings fescue; lane 4, E+ tall fescue; lane 5, Chewings fescue 1117 artificially infected with the Neotyphodium sp. Four micrograms of poly(A + ) RNA was used for each sample.
Figure Legend Snippet: RNA gel-blot analysis of β-1,6-glucanase transcripts. A, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, Neotyphodium sp. grown on 0.5% (w/v) pustulan; lane 4, Neotyphodium sp. grown on 0.5% (w/v) laminarin. Fifteen micrograms of total RNA was used from E− and E+ P. ampla leaf sheaths. Ten micrograms of total RNA was used from the Neotyphodium sp. fungal cultures. B, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, E+ Chewings fescue; lane 4, E+ tall fescue; lane 5, Chewings fescue 1117 artificially infected with the Neotyphodium sp. Four micrograms of poly(A + ) RNA was used for each sample.

Techniques Used: Western Blot, Infection

9) Product Images from "Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence"

Article Title: Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005392

Reactivity of the antiβ-glucan mAbs to β-glucans of different molecular structure. Panels A: dose-effect, ELISA mAb binding curves to plastic-adsorbed laminarin ( β1,3 glucan), pustulan (linear β1,6 glucan) and C. albicans β-glucan (mixed, highly branched β1,3/β1,6-glucan). The graph illustrates the outcome in a typical experiment out of five performed with similar results. Binding is expressed as mean O.D. 405 nm readings from triplicate wells after subtraction of O.D. from the negative controls (wells reacted with no mAb or with an irrelevant mAb). SEM values were always
Figure Legend Snippet: Reactivity of the antiβ-glucan mAbs to β-glucans of different molecular structure. Panels A: dose-effect, ELISA mAb binding curves to plastic-adsorbed laminarin ( β1,3 glucan), pustulan (linear β1,6 glucan) and C. albicans β-glucan (mixed, highly branched β1,3/β1,6-glucan). The graph illustrates the outcome in a typical experiment out of five performed with similar results. Binding is expressed as mean O.D. 405 nm readings from triplicate wells after subtraction of O.D. from the negative controls (wells reacted with no mAb or with an irrelevant mAb). SEM values were always

Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

Microarray analyses of the interactions of the mAbs with different gluco-oligosaccharide probes. The gluco-oligosaccharide probes were printed as duplicate spots and the binding was assayed with the IgG mAb at 0.1 µg/ml (Panel A) and the IgM mAb at 0.5 µg/ml (Panel B). Numerical scores are shown for the binding signals, means of duplicate values at 2 and 7 fmol/spot (blue and red bars, respectively, with error bars). The gluco-oligosaccharide probes tested included oligosaccharides from maltodextrins (α1–4), dextran (α1–6); curdlan (β1–3); cellulose (β1–4); and pustulan (β1–6). Numbers on the X axis indicate degree of polymerization (DP) of the major components in the oligosaccharide fractions.
Figure Legend Snippet: Microarray analyses of the interactions of the mAbs with different gluco-oligosaccharide probes. The gluco-oligosaccharide probes were printed as duplicate spots and the binding was assayed with the IgG mAb at 0.1 µg/ml (Panel A) and the IgM mAb at 0.5 µg/ml (Panel B). Numerical scores are shown for the binding signals, means of duplicate values at 2 and 7 fmol/spot (blue and red bars, respectively, with error bars). The gluco-oligosaccharide probes tested included oligosaccharides from maltodextrins (α1–4), dextran (α1–6); curdlan (β1–3); cellulose (β1–4); and pustulan (β1–6). Numbers on the X axis indicate degree of polymerization (DP) of the major components in the oligosaccharide fractions.

Techniques Used: Microarray, Binding Assay

10) Product Images from "A novel glyco-conjugate vaccine against fungal pathogens"

Article Title: A novel glyco-conjugate vaccine against fungal pathogens

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20050749

Vaccination with the Lam-CRM conjugate induces antibody-mediated anti- Candida protection in a murine experimental model of disseminated infection. (a) Anti-β-glucan IgG and IgM titers in Lam-CRM–vaccinated mice. The graph shows the ranges of ELISA titers against the indicated antigens measured in five groups of 6–12 mice (for a total of 46 animals) independently immunized with Lam-CRM. MP, mannoproteins; Pust, pustulan. (b) Survival rates of mice immunized with Lam or CRM or with the Lam-CRM conjugate, as compared with nonimmunized mice (Adj), after a lethal systemic challenge with C. albicans (cumulative data from three independent experiments and 28 mice per group). (c) Fungal burden in the kidneys from four Lam-CRM–vaccinated or four control CRM-vaccinated mice on day 2 after i.v. infection with C. albicans . (d) Number of fungal CFU in kidneys from naive mice given a single administration of anti-Lam-CRM, anti-CRM, or nonimmune (Adj) serum 2 h before an i.v. challenge with C. albicans . Data are from three independent experiments with a total of nine mice per group. (e) Reversal of the passive protection after serum adsorption with Candida cells. The experiment was performed with three mice per group. (f) Effect of the passive vaccination with Protein A affinity–separated fractions of the Lam-CRM serum on Candida kidney load. Data are from three mice per group. Some details on isotype and subclass of anti-β-glucan immunoglobulin in pool A, B and C are given in Fig. S2.
Figure Legend Snippet: Vaccination with the Lam-CRM conjugate induces antibody-mediated anti- Candida protection in a murine experimental model of disseminated infection. (a) Anti-β-glucan IgG and IgM titers in Lam-CRM–vaccinated mice. The graph shows the ranges of ELISA titers against the indicated antigens measured in five groups of 6–12 mice (for a total of 46 animals) independently immunized with Lam-CRM. MP, mannoproteins; Pust, pustulan. (b) Survival rates of mice immunized with Lam or CRM or with the Lam-CRM conjugate, as compared with nonimmunized mice (Adj), after a lethal systemic challenge with C. albicans (cumulative data from three independent experiments and 28 mice per group). (c) Fungal burden in the kidneys from four Lam-CRM–vaccinated or four control CRM-vaccinated mice on day 2 after i.v. infection with C. albicans . (d) Number of fungal CFU in kidneys from naive mice given a single administration of anti-Lam-CRM, anti-CRM, or nonimmune (Adj) serum 2 h before an i.v. challenge with C. albicans . Data are from three independent experiments with a total of nine mice per group. (e) Reversal of the passive protection after serum adsorption with Candida cells. The experiment was performed with three mice per group. (f) Effect of the passive vaccination with Protein A affinity–separated fractions of the Lam-CRM serum on Candida kidney load. Data are from three mice per group. Some details on isotype and subclass of anti-β-glucan immunoglobulin in pool A, B and C are given in Fig. S2.

Techniques Used: Laser Capture Microdissection, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Adsorption

11) Product Images from "A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi"

Article Title: A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121801

β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P
Figure Legend Snippet: β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P

Techniques Used: Staining, Expressing

Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.
Figure Legend Snippet: Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.

Techniques Used: Recombinant, Binding Assay, Dot Blot, Staining

V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. k a in 10 4 sec −1 M −1 , k d in 10 −5 sec −1 , K D in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants ( k a ) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.
Figure Legend Snippet: V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. k a in 10 4 sec −1 M −1 , k d in 10 −5 sec −1 , K D in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants ( k a ) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.

Techniques Used: SPR Assay, Binding Assay, Size-exclusion Chromatography, Molecular Weight

12) Product Images from "Adjuvanticity of a Recombinant Calreticulin Fragment in Assisting Anti-?-Glucan IgG Responses in T Cell-Deficient Mice"

Article Title: Adjuvanticity of a Recombinant Calreticulin Fragment in Assisting Anti-?-Glucan IgG Responses in T Cell-Deficient Mice

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00689-12

Enhanced immunogenicity of LAM-CRT and LAM-EGFP over unconjugated laminarin. Groups of BALB/c mice (6 per group) were s.c. injected with laminarin (A), LAM-EGFP (B), or LAM-CRT (C) or with rCRT/39–272 (F) (in PBS; 100 μg/mouse) and boosted with 50 μg of the same Ag preparations 2 weeks later. The mice were bled at different time points thereafter, and the combined sera (1/200 dilution) were assayed, in triplicate wells, for IgM and IgG using laminarin-based ELISAs. The detection antibody was HRP-conjugated goat anti-mouse IgM or IgG with OPD as the substrate. (D) Sera of the LAM-CRT group (mixture of equal proportions, 1/200 dilution), collected on day 28, were assayed in laminarin-based ELISAs for IgG subclasses using HRP-conjugated goat anti-mouse Abs against IgG1, IgG2a, IgG2b, or IgG3 for detection. (E) Antisera from all 3 groups (mixture of equal proportions for each group) were also titrated against laminarin using HRP-labeled IgG1-specific Abs for detection. (F) ELISAs were also carried out to test cross reactivity of antisera from the rCRT/39–272 group using microtiter plates precoated, in triplicate, with rCRT/39–272, rEGFP, pustulan, laminarin, mannan, alginic acid, dextran, and LPS. The results are expressed as mean OD 492 ± SD. *, P
Figure Legend Snippet: Enhanced immunogenicity of LAM-CRT and LAM-EGFP over unconjugated laminarin. Groups of BALB/c mice (6 per group) were s.c. injected with laminarin (A), LAM-EGFP (B), or LAM-CRT (C) or with rCRT/39–272 (F) (in PBS; 100 μg/mouse) and boosted with 50 μg of the same Ag preparations 2 weeks later. The mice were bled at different time points thereafter, and the combined sera (1/200 dilution) were assayed, in triplicate wells, for IgM and IgG using laminarin-based ELISAs. The detection antibody was HRP-conjugated goat anti-mouse IgM or IgG with OPD as the substrate. (D) Sera of the LAM-CRT group (mixture of equal proportions, 1/200 dilution), collected on day 28, were assayed in laminarin-based ELISAs for IgG subclasses using HRP-conjugated goat anti-mouse Abs against IgG1, IgG2a, IgG2b, or IgG3 for detection. (E) Antisera from all 3 groups (mixture of equal proportions for each group) were also titrated against laminarin using HRP-labeled IgG1-specific Abs for detection. (F) ELISAs were also carried out to test cross reactivity of antisera from the rCRT/39–272 group using microtiter plates precoated, in triplicate, with rCRT/39–272, rEGFP, pustulan, laminarin, mannan, alginic acid, dextran, and LPS. The results are expressed as mean OD 492 ± SD. *, P

Techniques Used: Laser Capture Microdissection, Mouse Assay, Injection, Labeling

Carbohydrate antigen specificity of LAM-CRT-induced Abs. Groups of BALB/c mice (A) and nude mice (B) were s.c. injected with LAM-CRT (in PBS; 100 μg/mouse, 6 per group) and boosted with 50 μg of the same Ag preparations 2 weeks later. Antisera collected on day 28 from each group were pooled, diluted 1/200, and dispensed in triplicate wells that had been precoated with dextran, pustulan, linear β-1,3-glucan, laminarin, mannan, alginic acid, and heparin for ELISAs. HRP-conjugated goat anti-mouse IgG was employed as a detection Ab followed by development with OPD. Wells not precoated with any Ags (None) were included as a control. The results are expressed as OD 492 with SD. ***, P
Figure Legend Snippet: Carbohydrate antigen specificity of LAM-CRT-induced Abs. Groups of BALB/c mice (A) and nude mice (B) were s.c. injected with LAM-CRT (in PBS; 100 μg/mouse, 6 per group) and boosted with 50 μg of the same Ag preparations 2 weeks later. Antisera collected on day 28 from each group were pooled, diluted 1/200, and dispensed in triplicate wells that had been precoated with dextran, pustulan, linear β-1,3-glucan, laminarin, mannan, alginic acid, and heparin for ELISAs. HRP-conjugated goat anti-mouse IgG was employed as a detection Ab followed by development with OPD. Wells not precoated with any Ags (None) were included as a control. The results are expressed as OD 492 with SD. ***, P

Techniques Used: Laser Capture Microdissection, Mouse Assay, Injection

13) Product Images from "Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin"

Article Title: Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

Journal: Eukaryotic Cell

doi: 10.1128/EC.00287-14

Competition of pustulan with the cell wall receptors for binding of K2 toxin in BY4741, Δ kre1 or Δ tok1 cells. Yeast cells (5 × 10 5 ) were incubated for 1 h with 9 mg of pustulan in the presence of 10 3 U/ml of K2 toxin. Surviving
Figure Legend Snippet: Competition of pustulan with the cell wall receptors for binding of K2 toxin in BY4741, Δ kre1 or Δ tok1 cells. Yeast cells (5 × 10 5 ) were incubated for 1 h with 9 mg of pustulan in the presence of 10 3 U/ml of K2 toxin. Surviving

Techniques Used: Binding Assay, Incubation

14) Product Images from "Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence"

Article Title: Protection by Anti-?-Glucan Antibodies Is Associated with Restricted ?-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005392

Reactivity of the antiβ-glucan mAbs to β-glucans of different molecular structure. Panels A: dose-effect, ELISA mAb binding curves to plastic-adsorbed laminarin ( β1,3 glucan), pustulan (linear β1,6 glucan) and C. albicans β-glucan (mixed, highly branched β1,3/β1,6-glucan). The graph illustrates the outcome in a typical experiment out of five performed with similar results. Binding is expressed as mean O.D. 405 nm readings from triplicate wells after subtraction of O.D. from the negative controls (wells reacted with no mAb or with an irrelevant mAb). SEM values were always
Figure Legend Snippet: Reactivity of the antiβ-glucan mAbs to β-glucans of different molecular structure. Panels A: dose-effect, ELISA mAb binding curves to plastic-adsorbed laminarin ( β1,3 glucan), pustulan (linear β1,6 glucan) and C. albicans β-glucan (mixed, highly branched β1,3/β1,6-glucan). The graph illustrates the outcome in a typical experiment out of five performed with similar results. Binding is expressed as mean O.D. 405 nm readings from triplicate wells after subtraction of O.D. from the negative controls (wells reacted with no mAb or with an irrelevant mAb). SEM values were always

Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

Microarray analyses of the interactions of the mAbs with different gluco-oligosaccharide probes. The gluco-oligosaccharide probes were printed as duplicate spots and the binding was assayed with the IgG mAb at 0.1 µg/ml (Panel A) and the IgM mAb at 0.5 µg/ml (Panel B). Numerical scores are shown for the binding signals, means of duplicate values at 2 and 7 fmol/spot (blue and red bars, respectively, with error bars). The gluco-oligosaccharide probes tested included oligosaccharides from maltodextrins (α1–4), dextran (α1–6); curdlan (β1–3); cellulose (β1–4); and pustulan (β1–6). Numbers on the X axis indicate degree of polymerization (DP) of the major components in the oligosaccharide fractions.
Figure Legend Snippet: Microarray analyses of the interactions of the mAbs with different gluco-oligosaccharide probes. The gluco-oligosaccharide probes were printed as duplicate spots and the binding was assayed with the IgG mAb at 0.1 µg/ml (Panel A) and the IgM mAb at 0.5 µg/ml (Panel B). Numerical scores are shown for the binding signals, means of duplicate values at 2 and 7 fmol/spot (blue and red bars, respectively, with error bars). The gluco-oligosaccharide probes tested included oligosaccharides from maltodextrins (α1–4), dextran (α1–6); curdlan (β1–3); cellulose (β1–4); and pustulan (β1–6). Numbers on the X axis indicate degree of polymerization (DP) of the major components in the oligosaccharide fractions.

Techniques Used: Microarray, Binding Assay

15) Product Images from "Development of a novel β-1,6-glucan–specific detection system using functionally-modified recombinant endo-β-1,6-glucanase"

Article Title: Development of a novel β-1,6-glucan–specific detection system using functionally-modified recombinant endo-β-1,6-glucanase

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA119.011851

β-1,6-Glucanase retains its ability to capture β-1,6-glucan after losing its glucan hydrolase activity via point mutations in the catalytic domain. A, direct binding activity of Neg1 variants to pustulan. The binding capacity of β-1,6-glucanase variants Neg1–E225Q, Neg1–E321Q, and Neg1–E225Q/E321Q to solid-phased laminarin ( red ) or pustulan ( blue ) was evaluated by a direct ELISA-like assay. B and C , affinities of Neg1 variants to pustulan. The kinetic binding level of Neg1 and its variants to the pustulan-conjugated spencer tip was monitored by the BLI method ( B ), and the K D value of E321Q–His was calculated with 2-fold serially-diluted probes ( C ). D, thermal stability of E321Q–His. The binding activity of heat-treated (range, 20–90 °C for 5 min) E321Q–His to pustulan was verified with a direct ELISA-like assay using pustulan-coated plates. E, pH stability of E321Q–His. E321Q–His diluted in various pH conditions with McIlvaine (range, pH 2.2–7.8, red ) or modified Britton-Robinson (range, pH 4–11, blue ) buffer was incubated with solid-phased pustulan, and the glucan-binding capacity of E321Q–His was evaluated by direct ELISA. F, effect of pH on the glucan-binding ability of E321Q–His during association or dissociation by the BLI method. For analyzing the association phase, the pustulan-conjugated spencer tip was incubated with E321Q–His in assay buffer (pH 4–11) regulated with modified Britton-Robinson, and dissociation data were collected with PBS ( left panel ). For analyzing the dissociation phase, the spencer tip was incubated with E321Q–His in PBS, and the dissociation data were collected with assay buffer (pH 4–11) regulated with modified Britton-Robinson ( right panel ). Representative graphs from at least two independent experiments per assay are shown.
Figure Legend Snippet: β-1,6-Glucanase retains its ability to capture β-1,6-glucan after losing its glucan hydrolase activity via point mutations in the catalytic domain. A, direct binding activity of Neg1 variants to pustulan. The binding capacity of β-1,6-glucanase variants Neg1–E225Q, Neg1–E321Q, and Neg1–E225Q/E321Q to solid-phased laminarin ( red ) or pustulan ( blue ) was evaluated by a direct ELISA-like assay. B and C , affinities of Neg1 variants to pustulan. The kinetic binding level of Neg1 and its variants to the pustulan-conjugated spencer tip was monitored by the BLI method ( B ), and the K D value of E321Q–His was calculated with 2-fold serially-diluted probes ( C ). D, thermal stability of E321Q–His. The binding activity of heat-treated (range, 20–90 °C for 5 min) E321Q–His to pustulan was verified with a direct ELISA-like assay using pustulan-coated plates. E, pH stability of E321Q–His. E321Q–His diluted in various pH conditions with McIlvaine (range, pH 2.2–7.8, red ) or modified Britton-Robinson (range, pH 4–11, blue ) buffer was incubated with solid-phased pustulan, and the glucan-binding capacity of E321Q–His was evaluated by direct ELISA. F, effect of pH on the glucan-binding ability of E321Q–His during association or dissociation by the BLI method. For analyzing the association phase, the pustulan-conjugated spencer tip was incubated with E321Q–His in assay buffer (pH 4–11) regulated with modified Britton-Robinson, and dissociation data were collected with PBS ( left panel ). For analyzing the dissociation phase, the spencer tip was incubated with E321Q–His in PBS, and the dissociation data were collected with assay buffer (pH 4–11) regulated with modified Britton-Robinson ( right panel ). Representative graphs from at least two independent experiments per assay are shown.

Techniques Used: Activity Assay, Binding Assay, Direct ELISA, Modification, Incubation

Application of sandwich ELISA using Neg1–E321Q for the quantification of naturally released β-1,6-glucan from C. albicans . A, standard curve of 3-fold serial dilutions of pustulan (concentration range, 30.5 pg/ml to 22.2 ng/ml). B, flowchart of the experiment for in vitro culture of C. albicans strain NBRC1385 and image of growth conformation. C, reactivity of sandwich ELISA to 3-fold serial dilutions of C. albicans strain NBRC1385 culture supernatant ( blue ) or Candida -free medium ( red ). β-1,6-Glucan ( D ) or β-1,3-glucan ( E ) content in culture supernatant of C. albicans strain NBRC1385 diluted 250- or 2,000-fold ( blue ) or Candida -free medium ( red ) is shown. F, blood clearance of β-1,6-glucan in mice. Serum was collected at 1, 10, and 30 min and at 24 h after intravenous injection of C. albicans NBRC1385 culture supernatant ( blue ) or Candida -free medium ( red ). The serum was diluted twice, and β-1,6-glucan concentrations were measured. G, images showing the proliferation of C. albicans strains BIG104 and BIG105. β-1,3-Glucan ( H ) or β-1,6-glucan ( I ) content in culture supernatants of C. albicans strains BIG104 and BIG105 or Candida -free medium is shown. Supernatants and blanks were diluted 100- and 50-fold for the β-1,6-glucan ELISA and LAL test, respectively. Data are presented as mean ± S.D. of values in duplicate ( A , C , D , and H ) or triplicate ( E and I ), or mean ± S.E. ( n = 3) ( F ). Shown are representative results from at least two independent experiments. Scale bars , 100 μm.
Figure Legend Snippet: Application of sandwich ELISA using Neg1–E321Q for the quantification of naturally released β-1,6-glucan from C. albicans . A, standard curve of 3-fold serial dilutions of pustulan (concentration range, 30.5 pg/ml to 22.2 ng/ml). B, flowchart of the experiment for in vitro culture of C. albicans strain NBRC1385 and image of growth conformation. C, reactivity of sandwich ELISA to 3-fold serial dilutions of C. albicans strain NBRC1385 culture supernatant ( blue ) or Candida -free medium ( red ). β-1,6-Glucan ( D ) or β-1,3-glucan ( E ) content in culture supernatant of C. albicans strain NBRC1385 diluted 250- or 2,000-fold ( blue ) or Candida -free medium ( red ) is shown. F, blood clearance of β-1,6-glucan in mice. Serum was collected at 1, 10, and 30 min and at 24 h after intravenous injection of C. albicans NBRC1385 culture supernatant ( blue ) or Candida -free medium ( red ). The serum was diluted twice, and β-1,6-glucan concentrations were measured. G, images showing the proliferation of C. albicans strains BIG104 and BIG105. β-1,3-Glucan ( H ) or β-1,6-glucan ( I ) content in culture supernatants of C. albicans strains BIG104 and BIG105 or Candida -free medium is shown. Supernatants and blanks were diluted 100- and 50-fold for the β-1,6-glucan ELISA and LAL test, respectively. Data are presented as mean ± S.D. of values in duplicate ( A , C , D , and H ) or triplicate ( E and I ), or mean ± S.E. ( n = 3) ( F ). Shown are representative results from at least two independent experiments. Scale bars , 100 μm.

Techniques Used: Sandwich ELISA, Concentration Assay, In Vitro, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

β-1,6-Glucan is produced and can be detected in C57BL/6 mice after systemic Candida infection. A, β-1,6-glucan production by C. albicans SC5314 in vitro . β-1,6-Glucan was measured in the C. albicans SC5314 culture supernatant after 24 h or in Candida -free medium as control, which were diluted 50-fold. An ELISA-like assay based on Neg1–E321Q with pustulan as the standard of β-1,6-glucan was used. B–F, β-1,6-glucan production by C. albicans SC5314 in vivo . Concentrations of β-1,6-glucan in serum ( B ), kidney ( C ), spleen ( D ), liver (E ), and brain ( F ) isolated from C57BL/6 mice on days 0, 3, 6, and 9 after C. albicans intravenous injection were measured by sandwich ELISA. Data are presented as mean ± S.E. ( n = 6–8 for serum and n = 4 for organ homogenates). Significant differences of days 3, 6, and 9 relative to day 0: *, p
Figure Legend Snippet: β-1,6-Glucan is produced and can be detected in C57BL/6 mice after systemic Candida infection. A, β-1,6-glucan production by C. albicans SC5314 in vitro . β-1,6-Glucan was measured in the C. albicans SC5314 culture supernatant after 24 h or in Candida -free medium as control, which were diluted 50-fold. An ELISA-like assay based on Neg1–E321Q with pustulan as the standard of β-1,6-glucan was used. B–F, β-1,6-glucan production by C. albicans SC5314 in vivo . Concentrations of β-1,6-glucan in serum ( B ), kidney ( C ), spleen ( D ), liver (E ), and brain ( F ) isolated from C57BL/6 mice on days 0, 3, 6, and 9 after C. albicans intravenous injection were measured by sandwich ELISA. Data are presented as mean ± S.E. ( n = 6–8 for serum and n = 4 for organ homogenates). Significant differences of days 3, 6, and 9 relative to day 0: *, p

Techniques Used: Produced, Mouse Assay, Infection, In Vitro, Enzyme-linked Immunosorbent Assay, In Vivo, Isolation, Injection, Sandwich ELISA

Neg1–E321Q recognizes β-1,6-glucan on the cell wall of both yeast and hyphae forms of C. albicans . A and B , direct binding of Neg1–E321Q–His (0–5 μg/ml) to the heat-killed yeast form of C. albicans ( HKCA ) was analyzed by FACS, and data are presented as representative histograms ( A ) and summary data of median fluorescence intensity ( B ). C, Structure-specific binding of Neg1–E321Q–His onto the yeast cell surface. HKCA was incubated with Neg1–E321Q–His in the presence of pustulan, laminarin, or mannan (0–100 μg/ml) and analyzed by FACS, and data are presented as representative histograms. D, Neg1–E321Q binds to the cell wall of the hyphal form of C. albicans . Fixed hyphae were stained with Neg1–E321Q–His and probes specific for β-1,3-glucan (dectin-1), mannan (ConA), and chitin (CFW). Shown are merged images from the four different probes that indicate localization. Differential interface contrast images are also shown. Scale bars , 50 μm. Shown are representative results from at least two independent experiments.
Figure Legend Snippet: Neg1–E321Q recognizes β-1,6-glucan on the cell wall of both yeast and hyphae forms of C. albicans . A and B , direct binding of Neg1–E321Q–His (0–5 μg/ml) to the heat-killed yeast form of C. albicans ( HKCA ) was analyzed by FACS, and data are presented as representative histograms ( A ) and summary data of median fluorescence intensity ( B ). C, Structure-specific binding of Neg1–E321Q–His onto the yeast cell surface. HKCA was incubated with Neg1–E321Q–His in the presence of pustulan, laminarin, or mannan (0–100 μg/ml) and analyzed by FACS, and data are presented as representative histograms. D, Neg1–E321Q binds to the cell wall of the hyphal form of C. albicans . Fixed hyphae were stained with Neg1–E321Q–His and probes specific for β-1,3-glucan (dectin-1), mannan (ConA), and chitin (CFW). Shown are merged images from the four different probes that indicate localization. Differential interface contrast images are also shown. Scale bars , 50 μm. Shown are representative results from at least two independent experiments.

Techniques Used: Binding Assay, FACS, Fluorescence, Incubation, Staining

Neg1–E321Q exerts structure-specific and molecular size-dependent ligand-binding activity. A, reactivity of Neg1–E321Q with various glucans. Pustulan-coated plates were incubated with Neg1–E321Q in the presence of various glucans (concentration, 20 or 100 μg/ml) or PBS as a control. Data shown as inhibition rates (%) were calculated with absorbance from PBS as 0 and blank well as 100 and represent the mean ± S.D. of values of duplicate analyses. B, direct ELISA using pustulan- or gentio-oligosaccharides (DP 2–6)-coated plates and Neg1–E321Q. C, competitive ELISA was employed to verify the interaction between Neg1–E321Q and low molecular weight β-1,6-glucan. A pustulan-coated plate was incubated with Neg1–E321Q in the presence of soluble pustulan or gentio-oligosaccharides (DP 2–6). D, schematic of the Neg1–E321Q–Nluc fusion protein. E, SDS-PAGE image of E321Q–Nluc. Purified recombinant Neg1–E321Q–Nluc and Neg1–E321Q were separated using 11% polyacrylamide gel, and bands were visualized by Coomassie Brilliant Blue. F, direct binding of Neg1–E321Q–Nluc to β-1,6-glucan. Pustulan (concentration, 0–5 μg/ml)-coated plates were blocked and incubated with Neg1–E321Q–Nluc (2 μg/ml) for 1 h, and the luciferase activity was measured. Data represent the mean ± S.D. of values of triplicate analyses. G, HPLC separation of hydrolyzed pustulan and visualized by FACE. Pustulan was hydrolyzed with hydrochloric acid, lyophilized, and dissolved in water. Samples were fractionated every minute, labeled with ANTS, and analyzed by FACE using 30% ( left panel ) and 40% ( right panel ) gel. ANTS-labeled hydrolyzed pustulan (before HPLC separation) is shown in the left margin as standards (indicated as P ). H, dot-blot analysis using Neg1–E321Q–Nluc. Each ANTS-labeled fraction of HPLC was spotted onto the membrane. The image was taken under UV light ( upper panel ) and by using a chemiluminescent scanner ( lower panel ). Number indicates each fraction. Blank sample (bromphenol blue) and ANTS-labeled hydrolyzed pustulan (before HPLC separation) were also spotted on the area labeled B and P , respectively. Shown are representative results from at least two independent experiments.
Figure Legend Snippet: Neg1–E321Q exerts structure-specific and molecular size-dependent ligand-binding activity. A, reactivity of Neg1–E321Q with various glucans. Pustulan-coated plates were incubated with Neg1–E321Q in the presence of various glucans (concentration, 20 or 100 μg/ml) or PBS as a control. Data shown as inhibition rates (%) were calculated with absorbance from PBS as 0 and blank well as 100 and represent the mean ± S.D. of values of duplicate analyses. B, direct ELISA using pustulan- or gentio-oligosaccharides (DP 2–6)-coated plates and Neg1–E321Q. C, competitive ELISA was employed to verify the interaction between Neg1–E321Q and low molecular weight β-1,6-glucan. A pustulan-coated plate was incubated with Neg1–E321Q in the presence of soluble pustulan or gentio-oligosaccharides (DP 2–6). D, schematic of the Neg1–E321Q–Nluc fusion protein. E, SDS-PAGE image of E321Q–Nluc. Purified recombinant Neg1–E321Q–Nluc and Neg1–E321Q were separated using 11% polyacrylamide gel, and bands were visualized by Coomassie Brilliant Blue. F, direct binding of Neg1–E321Q–Nluc to β-1,6-glucan. Pustulan (concentration, 0–5 μg/ml)-coated plates were blocked and incubated with Neg1–E321Q–Nluc (2 μg/ml) for 1 h, and the luciferase activity was measured. Data represent the mean ± S.D. of values of triplicate analyses. G, HPLC separation of hydrolyzed pustulan and visualized by FACE. Pustulan was hydrolyzed with hydrochloric acid, lyophilized, and dissolved in water. Samples were fractionated every minute, labeled with ANTS, and analyzed by FACE using 30% ( left panel ) and 40% ( right panel ) gel. ANTS-labeled hydrolyzed pustulan (before HPLC separation) is shown in the left margin as standards (indicated as P ). H, dot-blot analysis using Neg1–E321Q–Nluc. Each ANTS-labeled fraction of HPLC was spotted onto the membrane. The image was taken under UV light ( upper panel ) and by using a chemiluminescent scanner ( lower panel ). Number indicates each fraction. Blank sample (bromphenol blue) and ANTS-labeled hydrolyzed pustulan (before HPLC separation) were also spotted on the area labeled B and P , respectively. Shown are representative results from at least two independent experiments.

Techniques Used: Ligand Binding Assay, Activity Assay, Incubation, Concentration Assay, Inhibition, Direct ELISA, Competitive ELISA, Molecular Weight, SDS Page, Purification, Recombinant, Binding Assay, Luciferase, High Performance Liquid Chromatography, Labeling, Dot Blot

β-1,6-Glucan is produced by and can be detected in a large number of clinical isolates of all major Candida species. A, β-1,6-glucan contents in the culture supernatants of C. albicans and non- albicans Candida strains. Yeasts ( C. albicans, 132; C. glabrata, 35; C. dubliniensis, 15; C. parapsilosis, 11; C. krusei, 11; C. tropicalis, 9; C. auris, 11) isolated at the National Institutes of Health Clinical Center and provided from the CDC AR bank were cultured for 24 h, and 10-fold diluted supernatants were analyzed using the β-1,6-glucan ELISA. B, kinetic production of β-1,6-glucan from C. glabrata . 35 strains of C. glabrata were cultured for 24 or 72 h, and β-1,6-glucan in 10-fold diluted supernatants was measured. Bars are presented as mean of values. C. β-1,6-glucan on the cell surface of Candida yeasts. Representative clinical isolates of the corresponding Candida species were fixed and incubated with PBS or Neg1–E321Q–biotin in the presence or absence of pustulan. Yeasts were further stained with PBS (control) or streptavidin-PE ( SA-PE ) and analyzed using flow cytometry. Significant differences from blank ( A ) or between two groups ( B ): *, p
Figure Legend Snippet: β-1,6-Glucan is produced by and can be detected in a large number of clinical isolates of all major Candida species. A, β-1,6-glucan contents in the culture supernatants of C. albicans and non- albicans Candida strains. Yeasts ( C. albicans, 132; C. glabrata, 35; C. dubliniensis, 15; C. parapsilosis, 11; C. krusei, 11; C. tropicalis, 9; C. auris, 11) isolated at the National Institutes of Health Clinical Center and provided from the CDC AR bank were cultured for 24 h, and 10-fold diluted supernatants were analyzed using the β-1,6-glucan ELISA. B, kinetic production of β-1,6-glucan from C. glabrata . 35 strains of C. glabrata were cultured for 24 or 72 h, and β-1,6-glucan in 10-fold diluted supernatants was measured. Bars are presented as mean of values. C. β-1,6-glucan on the cell surface of Candida yeasts. Representative clinical isolates of the corresponding Candida species were fixed and incubated with PBS or Neg1–E321Q–biotin in the presence or absence of pustulan. Yeasts were further stained with PBS (control) or streptavidin-PE ( SA-PE ) and analyzed using flow cytometry. Significant differences from blank ( A ) or between two groups ( B ): *, p

Techniques Used: Produced, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Staining, Flow Cytometry

Correlation between the detected concentrations of β-1,3-glucan and β-1,6-glucan in the culture supernatants of C. albicans isolates. C. albicans yeasts obtained from NBRC ( n = 9, red ) or the Kyorin University Hospital ( n = 23, blue ) were cultured for 24 h in vitro, and the supernatant was diluted 10-fold. The β-glucan contents in the culture supernatants were measured by ELISA (for β-1,6-glucan) and the LAL test (for β-1,3-glucan). Pustulan and pachyman were used as the standard glucans for the ELISA and LAL test, respectively.
Figure Legend Snippet: Correlation between the detected concentrations of β-1,3-glucan and β-1,6-glucan in the culture supernatants of C. albicans isolates. C. albicans yeasts obtained from NBRC ( n = 9, red ) or the Kyorin University Hospital ( n = 23, blue ) were cultured for 24 h in vitro, and the supernatant was diluted 10-fold. The β-glucan contents in the culture supernatants were measured by ELISA (for β-1,6-glucan) and the LAL test (for β-1,3-glucan). Pustulan and pachyman were used as the standard glucans for the ELISA and LAL test, respectively.

Techniques Used: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay

16) Product Images from "A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi"

Article Title: A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121801

β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P
Figure Legend Snippet: β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P

Techniques Used: Staining, Expressing

Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.
Figure Legend Snippet: Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.

Techniques Used: Recombinant, Binding Assay, Dot Blot, Staining

V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. k a in 10 4 sec −1 M −1 , k d in 10 −5 sec −1 , K D in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants ( k a ) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.
Figure Legend Snippet: V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. k a in 10 4 sec −1 M −1 , k d in 10 −5 sec −1 , K D in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants ( k a ) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.

Techniques Used: SPR Assay, Binding Assay, Size-exclusion Chromatography, Molecular Weight

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    β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of <t>pustulan</t> (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P
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    β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi

    doi: 10.1084/jem.20121801

    Figure Lengend Snippet: β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19 + CD5 + cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19 + CD5 + Zymosan + cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7 –expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P

    Article Snippet: 4 × 105 cells were cultured in the presence of CD40L expressing 3T3 fibroblasts ( ) in wells coated with pustulan (EMD Millipore) or in the presence of biotinylated anti-kappa or anti-lambda F(ab′)2 (Southern Biotech) coupled to anti-biotin antibody-coated MicroBeads (Miltenyi Biotec; 40 µg/ml).

    Techniques: Staining, Expressing

    Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.

    Journal: The Journal of Experimental Medicine

    Article Title: A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi

    doi: 10.1084/jem.20121801

    Figure Lengend Snippet: Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.

    Article Snippet: 4 × 105 cells were cultured in the presence of CD40L expressing 3T3 fibroblasts ( ) in wells coated with pustulan (EMD Millipore) or in the presence of biotinylated anti-kappa or anti-lambda F(ab′)2 (Southern Biotech) coupled to anti-biotin antibody-coated MicroBeads (Miltenyi Biotec; 40 µg/ml).

    Techniques: Recombinant, Binding Assay, Dot Blot, Staining

    V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. k a in 10 4 sec −1 M −1 , k d in 10 −5 sec −1 , K D in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants ( k a ) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi

    doi: 10.1084/jem.20121801

    Figure Lengend Snippet: V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. k a in 10 4 sec −1 M −1 , k d in 10 −5 sec −1 , K D in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants ( k a ) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.

    Article Snippet: 4 × 105 cells were cultured in the presence of CD40L expressing 3T3 fibroblasts ( ) in wells coated with pustulan (EMD Millipore) or in the presence of biotinylated anti-kappa or anti-lambda F(ab′)2 (Southern Biotech) coupled to anti-biotin antibody-coated MicroBeads (Miltenyi Biotec; 40 µg/ml).

    Techniques: SPR Assay, Binding Assay, Size-exclusion Chromatography, Molecular Weight

    Hst 5 binds selectively to β-1,3-glucans in the Candida cell wall. Representative cell wall polysaccharides (1–16 mg ml −1 ) consisting of laminarin (β-1,3-glucan) (Lam, red squares), sialic acid (Sia, grey circles), mannans (Man, black diamonds), pustulan (β-1,6-glucan) (Pus, black squares) or untreated control (Con) were pre-incubated with BHst 5 (31 µM) then added to C. albicans cells for 1 h. Cell wall extracts were immunoblotted to detect BHst 5 (A) and quantified by dentistometry (B). Reduction in cell wall binding was accompanied by reduced candidacidal activity of Hst 5 when incubated with the same polysaccharides in a dose-dependent manner (C). The same polysaccharides were coupled to Sepharose beads and percent binding of BHst 5 was assessed by elution affinity chromatography (D). Only laminarin was found to have significant binding with Hst 5. PCW and laminarinase-treated PCW were incubated with Hst 5, and unbound Hst 5 in supernatant (S) and bound Hst 5 in pellets (P) were detected on Tricine-SDS gels (E). Laminarinase digestion of PCW prevented Hst 5 binding (E). Cell wall binding of Hst 5 was reduced in a dose-dependent manner by laminarinase digests (F), when laminarinase digests (2–8 mg ml −1 ) were pre-incubated with Hst 5, then added to C. albicans cells. Killing activities of Hst 5 were decreased against C. albicans cells grown in YPD broth with caspofungin (1 or 2 ng ml −1 ) (G).

    Journal: Molecular Microbiology

    Article Title: Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans

    doi: 10.1111/j.1365-2958.2010.07210.x

    Figure Lengend Snippet: Hst 5 binds selectively to β-1,3-glucans in the Candida cell wall. Representative cell wall polysaccharides (1–16 mg ml −1 ) consisting of laminarin (β-1,3-glucan) (Lam, red squares), sialic acid (Sia, grey circles), mannans (Man, black diamonds), pustulan (β-1,6-glucan) (Pus, black squares) or untreated control (Con) were pre-incubated with BHst 5 (31 µM) then added to C. albicans cells for 1 h. Cell wall extracts were immunoblotted to detect BHst 5 (A) and quantified by dentistometry (B). Reduction in cell wall binding was accompanied by reduced candidacidal activity of Hst 5 when incubated with the same polysaccharides in a dose-dependent manner (C). The same polysaccharides were coupled to Sepharose beads and percent binding of BHst 5 was assessed by elution affinity chromatography (D). Only laminarin was found to have significant binding with Hst 5. PCW and laminarinase-treated PCW were incubated with Hst 5, and unbound Hst 5 in supernatant (S) and bound Hst 5 in pellets (P) were detected on Tricine-SDS gels (E). Laminarinase digestion of PCW prevented Hst 5 binding (E). Cell wall binding of Hst 5 was reduced in a dose-dependent manner by laminarinase digests (F), when laminarinase digests (2–8 mg ml −1 ) were pre-incubated with Hst 5, then added to C. albicans cells. Killing activities of Hst 5 were decreased against C. albicans cells grown in YPD broth with caspofungin (1 or 2 ng ml −1 ) (G).

    Article Snippet: BHst 5 (20 µl) or Hst 5 (20 µl) (final concentration = 31 µM) was added to 80 µl of each polysaccharide [1.125–20 mg ml−1 in 10 mM sodium phosphate buffer, pH 7.4 (NaPB)] including laminarin (β-1,3-glucan polymer; Sigma), sialic acid (Sigma), pustulan (β-1,6-glucan polymer; Calbiochem) or mannan (mannose polymer; Sigma) and incubated with C. albicans cells for 1 h at 30°C.

    Techniques: Laser Capture Microdissection, Incubation, Binding Assay, Activity Assay, Affinity Chromatography

    Glucosyl polysaccharides laminarin and pustulan. The carbon atom numbering schemes for each are indicated.

    Journal: Infection and Immunity

    Article Title: Interactions of Surfactant Proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus

    doi: 10.1128/IAI.69.4.2037-2044.2001

    Figure Lengend Snippet: Glucosyl polysaccharides laminarin and pustulan. The carbon atom numbering schemes for each are indicated.

    Article Snippet: Biological (Swampscott, Mass.), peptide N -glycosidase F (PNGase F) was purchased from New England Biolabs (Beverly, Mass.), pustulan was purchased from Calbiochem (San Diego, Calif.), and laminarin was purchased from Fluka (Buchs, Switzerland).

    Techniques:

    Inhibition of SP-D-induced yeast aggregation. Yeast cells were suspended in calcium-containing buffer with or without carbohydrate inhibitor at room temperature. After 5 min, recombinant human SP-D was added to all samples except the negative control (final SP-D concentration was 5 μg/ml). Buffer was added to the negative control sample. The A 700 of the suspensions was monitored every minute for 2 h after protein addition. For the graphs shown, the starting A 700 for all samples was normalized to the buffer control for ease of interpretation. Maltose, pustulan, and laminarin concentrations are reported as glucose equivalents (Glc eq.). The graph shows representative data from duplicate experiments.

    Journal: Infection and Immunity

    Article Title: Interactions of Surfactant Proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus

    doi: 10.1128/IAI.69.4.2037-2044.2001

    Figure Lengend Snippet: Inhibition of SP-D-induced yeast aggregation. Yeast cells were suspended in calcium-containing buffer with or without carbohydrate inhibitor at room temperature. After 5 min, recombinant human SP-D was added to all samples except the negative control (final SP-D concentration was 5 μg/ml). Buffer was added to the negative control sample. The A 700 of the suspensions was monitored every minute for 2 h after protein addition. For the graphs shown, the starting A 700 for all samples was normalized to the buffer control for ease of interpretation. Maltose, pustulan, and laminarin concentrations are reported as glucose equivalents (Glc eq.). The graph shows representative data from duplicate experiments.

    Article Snippet: Biological (Swampscott, Mass.), peptide N -glycosidase F (PNGase F) was purchased from New England Biolabs (Beverly, Mass.), pustulan was purchased from Calbiochem (San Diego, Calif.), and laminarin was purchased from Fluka (Buchs, Switzerland).

    Techniques: Inhibition, Recombinant, Negative Control, Concentration Assay, Gas Chromatography

    RNA gel-blot analysis of β-1,6-glucanase transcripts. A, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, Neotyphodium sp. grown on 0.5% (w/v) pustulan; lane 4, Neotyphodium sp. grown on 0.5% (w/v) laminarin. Fifteen micrograms of total RNA was used from E− and E+ P. ampla leaf sheaths. Ten micrograms of total RNA was used from the Neotyphodium sp. fungal cultures. B, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, E+ Chewings fescue; lane 4, E+ tall fescue; lane 5, Chewings fescue 1117 artificially infected with the Neotyphodium sp. Four micrograms of poly(A + ) RNA was used for each sample.

    Journal: Plant Physiology

    Article Title: Endophytic Fungal ?-1,6-Glucanase Expression in the Infected Host Grass 1

    doi: 10.1104/pp.010108

    Figure Lengend Snippet: RNA gel-blot analysis of β-1,6-glucanase transcripts. A, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, Neotyphodium sp. grown on 0.5% (w/v) pustulan; lane 4, Neotyphodium sp. grown on 0.5% (w/v) laminarin. Fifteen micrograms of total RNA was used from E− and E+ P. ampla leaf sheaths. Ten micrograms of total RNA was used from the Neotyphodium sp. fungal cultures. B, Lane 1, E− P. ampla ; lane 2, E+ P. ampla ; lane 3, E+ Chewings fescue; lane 4, E+ tall fescue; lane 5, Chewings fescue 1117 artificially infected with the Neotyphodium sp. Four micrograms of poly(A + ) RNA was used for each sample.

    Article Snippet: For growth on pustulan or laminarin, the endophyte was grown on plates of a minimal salts medium (M9; ) without Glc but supplemented with 0.25% (w/v) yeast extract and either 0.5% (w/v) pustulan (Calbiochem, La Jolla, CA) or 0.5% (w/v) laminarin (Sigma-Aldrich, St. Louis).

    Techniques: Western Blot, Infection