zymolyase  (Zymo Research)


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    Name:
    Zymolyase
    Description:
    Digestion of yeast and fungal cell walls is necessary for many experimental procedures including spheroplasting immunofluorescence transformation protein purification and others The use of lytic enzymes like Zymolyase is routinely used for digestion The Zymolyase from Zymo Research is prepared from Arthrobacter luteus lyophilized and packaged with a resuspension buffer The buffer has been optimized to confer maximal levels of enzymatic activity The main activities of the enzyme are β 1 3 glucanase and β 1 3 glucan laminaripentao hydrolase which hydrolyze glucose polymers at the β 1 3 glucan linkages releasing laminaripentaose as the principal product Optimal Zymolyase activity is at 30° 37°C lytic activity ceases at higher temperatures R Zymolyase includes 0 5 U µl RNase A when reconstituted Susceptible fungal genera Asbya Candida Debaryomyces Eremothecium Endomyces Hansenula Hanseniaspora Kloekera Kluyveromyces Lipomyces Metschikowia Pichia Pullularia Saccharomyces Saccharomyces Saccharomycopsis Schizosaccahromyces Torulopsis
    Catalog Number:
    e1004
    Price:
    None
    Applications:
    Yeast Lysis, Yeast Purification
    Size:
    1000 units
    Category:
    Life Science Reagents and Media
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    Structured Review

    Zymo Research zymolyase
    Zymolyase
    Digestion of yeast and fungal cell walls is necessary for many experimental procedures including spheroplasting immunofluorescence transformation protein purification and others The use of lytic enzymes like Zymolyase is routinely used for digestion The Zymolyase from Zymo Research is prepared from Arthrobacter luteus lyophilized and packaged with a resuspension buffer The buffer has been optimized to confer maximal levels of enzymatic activity The main activities of the enzyme are β 1 3 glucanase and β 1 3 glucan laminaripentao hydrolase which hydrolyze glucose polymers at the β 1 3 glucan linkages releasing laminaripentaose as the principal product Optimal Zymolyase activity is at 30° 37°C lytic activity ceases at higher temperatures R Zymolyase includes 0 5 U µl RNase A when reconstituted Susceptible fungal genera Asbya Candida Debaryomyces Eremothecium Endomyces Hansenula Hanseniaspora Kloekera Kluyveromyces Lipomyces Metschikowia Pichia Pullularia Saccharomyces Saccharomyces Saccharomycopsis Schizosaccahromyces Torulopsis
    https://www.bioz.com/result/zymolyase/product/Zymo Research
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    zymolyase - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection"

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.810440

    [ 14 C]Lysine cross-linked by TGase activity was found in low- and high-molecular-weight cell wall proteins and inhibition of TGase enzyme by cystamine and MDC induced changes in cell wall that sensitized cells to zymolyase treatment. A , paper chromatography of [ 14 C]putrescine-labeled material performed by C. albicans TGase activity and released from CW by zymolyase ( filled circles ). Solubilized fraction containing 20,000 cpm analyzed by paper chromatography was described under the “Experimental procedures.” [ 14 C]Putrescine substrate was also analyzed as a control ( open circles ). B , transglutaminase-mediated incorporation of [ 14 C]lysine into endogenous cell wall proteins. Reaction mixtures containing similar aliquots of cell walls and 2.5 μCi of [ 14 C]lysine with no cystamine ( open circles ) or with 50 m m cystamine ( filled circles ), boiled enzymatic source ( open triangles ), or 2 m m EDTA ( filled triangles ) were incubated for the indicated times; radioactivity in TCA-precipitable material was quantified. C , labeled samples with [ 14 C]lysine were sequentially released from cell walls by SDS, zymolyase, and chitinase and analyzed by 10% SDS-PAGE and fluorography. 10,000 cpm of labeled fractions were loaded in each lane. Lane 1, SDS-released material; lane 2 , zymolyase-released material; lane 3 , chitinase-released material. D and E, inhibition of TGase by cystamine and MDC increased sensitivity of C. albicans cells to treatment with zymolyase. Cells of C. albicans (adjusted to OD 600 nm = 0.5) previously incubated without ( circles ) or with 100 m m cystamine ( D ) or 3.5 m m MDC ( E ) for 1.5 h ( triangles ), 2 h ( inverted triangles ), and 4 h ( squares ) were treated with 50 μg ml −1 zymolyase 20T for up to 120 min. At the indicated times, OD 600 nm of cultures was monitored.
    Figure Legend Snippet: [ 14 C]Lysine cross-linked by TGase activity was found in low- and high-molecular-weight cell wall proteins and inhibition of TGase enzyme by cystamine and MDC induced changes in cell wall that sensitized cells to zymolyase treatment. A , paper chromatography of [ 14 C]putrescine-labeled material performed by C. albicans TGase activity and released from CW by zymolyase ( filled circles ). Solubilized fraction containing 20,000 cpm analyzed by paper chromatography was described under the “Experimental procedures.” [ 14 C]Putrescine substrate was also analyzed as a control ( open circles ). B , transglutaminase-mediated incorporation of [ 14 C]lysine into endogenous cell wall proteins. Reaction mixtures containing similar aliquots of cell walls and 2.5 μCi of [ 14 C]lysine with no cystamine ( open circles ) or with 50 m m cystamine ( filled circles ), boiled enzymatic source ( open triangles ), or 2 m m EDTA ( filled triangles ) were incubated for the indicated times; radioactivity in TCA-precipitable material was quantified. C , labeled samples with [ 14 C]lysine were sequentially released from cell walls by SDS, zymolyase, and chitinase and analyzed by 10% SDS-PAGE and fluorography. 10,000 cpm of labeled fractions were loaded in each lane. Lane 1, SDS-released material; lane 2 , zymolyase-released material; lane 3 , chitinase-released material. D and E, inhibition of TGase by cystamine and MDC increased sensitivity of C. albicans cells to treatment with zymolyase. Cells of C. albicans (adjusted to OD 600 nm = 0.5) previously incubated without ( circles ) or with 100 m m cystamine ( D ) or 3.5 m m MDC ( E ) for 1.5 h ( triangles ), 2 h ( inverted triangles ), and 4 h ( squares ) were treated with 50 μg ml −1 zymolyase 20T for up to 120 min. At the indicated times, OD 600 nm of cultures was monitored.

    Techniques Used: Activity Assay, Molecular Weight, Inhibition, Paper Chromatography, Labeling, Incubation, Radioactivity, SDS Page

    2) Product Images from "Antigen Release Kinetics in the Phagosome Are Critical to Cross-Presentation Efficiency"

    Article Title: Antigen Release Kinetics in the Phagosome Are Critical to Cross-Presentation Efficiency

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi:

    Ag displayed on the surface of yeast is cross-presented more efficiently than Ag expressed intracellularly in yeast. A , Diagram of the yeast surface display system of strain EBYN9V. The N9V epitope is in bold. B , Dose response of EBYN9V on cross-presentation. EBYN9V and wild-type EBY100 yeast were added to DCs at the indicated ratios. After 24 h, the DCs were assayed for the ability to stimulate IFN- γ secretion in cocultured N9V-specific T cells. Error bars, SDs of duplicate wells. C , EBYN9V yeast and yeast expressing the same Aga2p-N9V fusion protein intracellularly were added to DCs at a 20:1 ratio and tested for the ability to stimulate IFN- γ secretion in cocultured N9V-specific T cells. Lactacystin (5 μ M) or chloroquine (25 μ M) were added to some wells an hour before the yeast were introduced. Error bars, SDs of duplicate wells. D , Samples of the two yeast cultures and wt yeast were subjected to extensive reduction to release proteins disulfide-bonded to the cell wall. Subsequently, the yeast were treated with Zymolyase and lysed. The proteins reduced off the cell wall and the lysed cell extracts were slot-blotted onto the same nitrocellulose membrane and labeled for c- myc .
    Figure Legend Snippet: Ag displayed on the surface of yeast is cross-presented more efficiently than Ag expressed intracellularly in yeast. A , Diagram of the yeast surface display system of strain EBYN9V. The N9V epitope is in bold. B , Dose response of EBYN9V on cross-presentation. EBYN9V and wild-type EBY100 yeast were added to DCs at the indicated ratios. After 24 h, the DCs were assayed for the ability to stimulate IFN- γ secretion in cocultured N9V-specific T cells. Error bars, SDs of duplicate wells. C , EBYN9V yeast and yeast expressing the same Aga2p-N9V fusion protein intracellularly were added to DCs at a 20:1 ratio and tested for the ability to stimulate IFN- γ secretion in cocultured N9V-specific T cells. Lactacystin (5 μ M) or chloroquine (25 μ M) were added to some wells an hour before the yeast were introduced. Error bars, SDs of duplicate wells. D , Samples of the two yeast cultures and wt yeast were subjected to extensive reduction to release proteins disulfide-bonded to the cell wall. Subsequently, the yeast were treated with Zymolyase and lysed. The proteins reduced off the cell wall and the lysed cell extracts were slot-blotted onto the same nitrocellulose membrane and labeled for c- myc .

    Techniques Used: Expressing, Labeling

    3) Product Images from "Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis"

    Article Title: Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.1005940

    Effect of mechanical feedback strength and turgor pressure on cell viability. The strength of mechanical feedback, Γ, is experimentally varied by deleting MID2 and WSC1 . The dimensionless parameter ( Pρ w λ X )/(12 μ 0 m w ρ 0 k p ) is varied by changing the osmolarity of the external medium through dilution of the yeast growth media, YPD, in deionized H 2 O, effectively increasing turgor pressure P in cells. Cell lysis was measured using the PI staining viability assay ( Methods ). (A) Percent of lysed cells in the absence of α -factor for WT, mid2Δ and wsc1Δ mutants, as well as the mid2 Δ wsc1Δ double mutant. (B) Percent of WT lysed cells when grown in the presence of α -factor in YPD medium with decreasing osmolarity. (C) Percent of mid2Δ wsc1Δ lysed cells when grown both in the presence and absence of α -factor in YPD, in osmotically supported conditions (YPD + 1M sorbitol), as well as in hypo-osmotic conditions (100% H 2 O). (D) Percent of mid2Δ wsc1Δ lysed cells when grown in the presence of α -factor and osmotically supported media (YPD + sorbitol), diluted for decreasing osmolarities. (E) Percent of lysed cells in mid2Δ and wsc1Δ mutants, as well as the mid2Δ wsc1Δ double mutant, when grown in the presence of α -factor in YPD. (F) Theoretically predicted dynamical regimes for varying values of the mechanical feedback strength Γ and the ratio ( Pρ w λ X )/(12 μ 0 m w ρ 0 k p ). Decreasing osmolarity experimentally, corresponds to increasing P and, therefore, moving along horizontal lines in the positive direction. Addition of zymolyase, a cell wall degrading enzyme, corresponds to decreasing the cell wall viscosity, moving also along horizontal lines in the positive direction. (G) Images (DIC, PI staining and merge) showing the moments before and after the piercing of the cell wall at the tip of a mating projection and subsequent cell lysis of a mid2Δ cell after the addition of zymolyase, for video see S1 Video . Scale bar, 2 μm . (H) Temporal increase in the fraction of pierced mating projections for both mid2Δ (squares) and WT (circles) cells after addition of zymolyase.
    Figure Legend Snippet: Effect of mechanical feedback strength and turgor pressure on cell viability. The strength of mechanical feedback, Γ, is experimentally varied by deleting MID2 and WSC1 . The dimensionless parameter ( Pρ w λ X )/(12 μ 0 m w ρ 0 k p ) is varied by changing the osmolarity of the external medium through dilution of the yeast growth media, YPD, in deionized H 2 O, effectively increasing turgor pressure P in cells. Cell lysis was measured using the PI staining viability assay ( Methods ). (A) Percent of lysed cells in the absence of α -factor for WT, mid2Δ and wsc1Δ mutants, as well as the mid2 Δ wsc1Δ double mutant. (B) Percent of WT lysed cells when grown in the presence of α -factor in YPD medium with decreasing osmolarity. (C) Percent of mid2Δ wsc1Δ lysed cells when grown both in the presence and absence of α -factor in YPD, in osmotically supported conditions (YPD + 1M sorbitol), as well as in hypo-osmotic conditions (100% H 2 O). (D) Percent of mid2Δ wsc1Δ lysed cells when grown in the presence of α -factor and osmotically supported media (YPD + sorbitol), diluted for decreasing osmolarities. (E) Percent of lysed cells in mid2Δ and wsc1Δ mutants, as well as the mid2Δ wsc1Δ double mutant, when grown in the presence of α -factor in YPD. (F) Theoretically predicted dynamical regimes for varying values of the mechanical feedback strength Γ and the ratio ( Pρ w λ X )/(12 μ 0 m w ρ 0 k p ). Decreasing osmolarity experimentally, corresponds to increasing P and, therefore, moving along horizontal lines in the positive direction. Addition of zymolyase, a cell wall degrading enzyme, corresponds to decreasing the cell wall viscosity, moving also along horizontal lines in the positive direction. (G) Images (DIC, PI staining and merge) showing the moments before and after the piercing of the cell wall at the tip of a mating projection and subsequent cell lysis of a mid2Δ cell after the addition of zymolyase, for video see S1 Video . Scale bar, 2 μm . (H) Temporal increase in the fraction of pierced mating projections for both mid2Δ (squares) and WT (circles) cells after addition of zymolyase.

    Techniques Used: Lysis, Staining, Viability Assay, Mutagenesis

    4) Product Images from "Inhibition of Cdc42 during mitotic exit is required for cytokinesis"

    Article Title: Inhibition of Cdc42 during mitotic exit is required for cytokinesis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201301090

    Active Cdc42 interferes with cytokinesis and cell separation. (A) Cells transformed with vector (VEC) or GAL1-HA-CDC42 plasmids were spotted in fivefold dilutions on the indicated media (3 d at 25°C). OE, overexpression; GAL, galactose. (B) Expression of cdc42 G60D from the CDC42 promoter on a CEN plasmid (one to three copies per cell) is toxic to myo1Δ cells. Cells were grown on the indicated media (3 d at 25°C). Synthetic lethality is detected on 5-fluoroorotic acid (5FOA) media as a counterselection for the indicated URA3 -containing plasmids. (C) myo1Δ cells transformed with control or CDC24 2μ plasmids were grown on plates as in B. SC, synthetic complete. (D) Wild-type (wt) and bem2-ts cells were arrested in metaphase by CDC20 depletion, shifted to the restrictive temperature (37°C at 1.5 h), and processed for Cdc42 activation. Graph shows means ± SEM from three experiments. (E) Cells were arrested in metaphase, shifted to 37°C for 1 h, and released at 37°C. The percentage of cells with the indicated morphology is plotted as means ± SEM from three experiments. Connected indicates two adjacent cell bodies showing evidence of rebudding or repolarization. (F) Bright-field image of cells 50 min after release. Bar, 15 µm. (G) Cells from E were treated with zymolyase to digest the cell wall, and the percentage of large-budded or connected cells was scored. (H) Wild-type cells have a PS (arrow) sandwiched by secondary septa; bem2-ts cells have misaligned, multiple, or otherwise abnormal PSs (arrowheads). Bars, 500 nm.
    Figure Legend Snippet: Active Cdc42 interferes with cytokinesis and cell separation. (A) Cells transformed with vector (VEC) or GAL1-HA-CDC42 plasmids were spotted in fivefold dilutions on the indicated media (3 d at 25°C). OE, overexpression; GAL, galactose. (B) Expression of cdc42 G60D from the CDC42 promoter on a CEN plasmid (one to three copies per cell) is toxic to myo1Δ cells. Cells were grown on the indicated media (3 d at 25°C). Synthetic lethality is detected on 5-fluoroorotic acid (5FOA) media as a counterselection for the indicated URA3 -containing plasmids. (C) myo1Δ cells transformed with control or CDC24 2μ plasmids were grown on plates as in B. SC, synthetic complete. (D) Wild-type (wt) and bem2-ts cells were arrested in metaphase by CDC20 depletion, shifted to the restrictive temperature (37°C at 1.5 h), and processed for Cdc42 activation. Graph shows means ± SEM from three experiments. (E) Cells were arrested in metaphase, shifted to 37°C for 1 h, and released at 37°C. The percentage of cells with the indicated morphology is plotted as means ± SEM from three experiments. Connected indicates two adjacent cell bodies showing evidence of rebudding or repolarization. (F) Bright-field image of cells 50 min after release. Bar, 15 µm. (G) Cells from E were treated with zymolyase to digest the cell wall, and the percentage of large-budded or connected cells was scored. (H) Wild-type cells have a PS (arrow) sandwiched by secondary septa; bem2-ts cells have misaligned, multiple, or otherwise abnormal PSs (arrowheads). Bars, 500 nm.

    Techniques Used: Transformation Assay, Plasmid Preparation, Over Expression, Expressing, Activation Assay

    Related Articles

    Protease Inhibitor:

    Article Title: Antigen Release Kinetics in the Phagosome Are Critical to Cross-Presentation Efficiency
    Article Snippet: .. The yeast pellets were then washed with spheroplast buffer (50 mM Tris-HCl, pH 7.5, 1.4 M sorbitol, and 40 mM 2-ME), incubated with 2.4 U Zymolyase (Zymo Research) in 120 μ l spheroplast buffer containing a protease inhibitor mixture (Roche) for 15 min at 37°C, and boiled in 2% SDS for 5 min. .. The protein extracts were blotted onto nitrocellulose membrane with a slot-blotting apparatus (Bio-Rad).

    Incubation:

    Article Title: Antigen Release Kinetics in the Phagosome Are Critical to Cross-Presentation Efficiency
    Article Snippet: .. The yeast pellets were then washed with spheroplast buffer (50 mM Tris-HCl, pH 7.5, 1.4 M sorbitol, and 40 mM 2-ME), incubated with 2.4 U Zymolyase (Zymo Research) in 120 μ l spheroplast buffer containing a protease inhibitor mixture (Roche) for 15 min at 37°C, and boiled in 2% SDS for 5 min. .. The protein extracts were blotted onto nitrocellulose membrane with a slot-blotting apparatus (Bio-Rad).

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    Zymo Research zymolyase 100t
    Characterization of putative target genes of Prz1 implicated in cell-wall-related processes. (A) The heat map shows relative expression and Prz1 promoter occupancy for 15 putative target genes annotated to function in cell-wall organization or biogenesis. The color bars indicate relative expression and ChIP enrichment ratios between experimental and control strains. All microarray expression and ChIP-chip experiments were performed in replicate with dye reversal. (B) Cell-wall degradation assays. Wild-type and ∆ prz1 strains were grown in liquid YES medium, while nmt1 -driven prz1 + or empty vector (EV) were grown in liquid EMM minus thiamine for 18–24 hr. The samples were adjusted to matching cell densities and transferred to test tubes in the presence of 25 U/ml <t>Zymolyase</t> <t>100T.</t> The samples were shaken at 37°, and OD 600 readings were taken every 15 min to assess the degree of cell-wall degradation. Overexpression of prz1 + caused resistance to the cell-wall-degrading enzyme zymolyase ( P = 1.0e −4 ) while Δprz1 cells did not show significant sensitivity to zymolyase compared to wild type. (C) Spot dilution for micafungin sensitivity of deletion strains of the putative Prz1 target genes involved in cell-wall-related processes. Exponentially growing wild-type and deletion strains were pinned on solid YES medium containing 0.5 µg/ml micafungin and incubated at 30° for 3 days. (D) Cell-wall degradation assays. The Δomh1 strain was more sensitive to zymolyase treatment than wild type ( P = 1.6e −2 ). The zymolyase-resistant phenotype from overexpression of prz1 + was abrogated by loss of omh1 + ( P = 5.0e −4 ). The zymolyase experiments were repeated in triplicate, and error bars represent the standard error. The P -values were determined with ANOVA followed by a two-tailed t -test after 2 hr of zymolyase treatment.
    Zymolyase 100t, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/zymolyase 100t/product/Zymo Research
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    zymolyase 100t - by Bioz Stars, 2020-10
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    Characterization of putative target genes of Prz1 implicated in cell-wall-related processes. (A) The heat map shows relative expression and Prz1 promoter occupancy for 15 putative target genes annotated to function in cell-wall organization or biogenesis. The color bars indicate relative expression and ChIP enrichment ratios between experimental and control strains. All microarray expression and ChIP-chip experiments were performed in replicate with dye reversal. (B) Cell-wall degradation assays. Wild-type and ∆ prz1 strains were grown in liquid YES medium, while nmt1 -driven prz1 + or empty vector (EV) were grown in liquid EMM minus thiamine for 18–24 hr. The samples were adjusted to matching cell densities and transferred to test tubes in the presence of 25 U/ml Zymolyase 100T. The samples were shaken at 37°, and OD 600 readings were taken every 15 min to assess the degree of cell-wall degradation. Overexpression of prz1 + caused resistance to the cell-wall-degrading enzyme zymolyase ( P = 1.0e −4 ) while Δprz1 cells did not show significant sensitivity to zymolyase compared to wild type. (C) Spot dilution for micafungin sensitivity of deletion strains of the putative Prz1 target genes involved in cell-wall-related processes. Exponentially growing wild-type and deletion strains were pinned on solid YES medium containing 0.5 µg/ml micafungin and incubated at 30° for 3 days. (D) Cell-wall degradation assays. The Δomh1 strain was more sensitive to zymolyase treatment than wild type ( P = 1.6e −2 ). The zymolyase-resistant phenotype from overexpression of prz1 + was abrogated by loss of omh1 + ( P = 5.0e −4 ). The zymolyase experiments were repeated in triplicate, and error bars represent the standard error. The P -values were determined with ANOVA followed by a two-tailed t -test after 2 hr of zymolyase treatment.

    Journal: Genetics

    Article Title: Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast

    doi: 10.1534/genetics.115.184218

    Figure Lengend Snippet: Characterization of putative target genes of Prz1 implicated in cell-wall-related processes. (A) The heat map shows relative expression and Prz1 promoter occupancy for 15 putative target genes annotated to function in cell-wall organization or biogenesis. The color bars indicate relative expression and ChIP enrichment ratios between experimental and control strains. All microarray expression and ChIP-chip experiments were performed in replicate with dye reversal. (B) Cell-wall degradation assays. Wild-type and ∆ prz1 strains were grown in liquid YES medium, while nmt1 -driven prz1 + or empty vector (EV) were grown in liquid EMM minus thiamine for 18–24 hr. The samples were adjusted to matching cell densities and transferred to test tubes in the presence of 25 U/ml Zymolyase 100T. The samples were shaken at 37°, and OD 600 readings were taken every 15 min to assess the degree of cell-wall degradation. Overexpression of prz1 + caused resistance to the cell-wall-degrading enzyme zymolyase ( P = 1.0e −4 ) while Δprz1 cells did not show significant sensitivity to zymolyase compared to wild type. (C) Spot dilution for micafungin sensitivity of deletion strains of the putative Prz1 target genes involved in cell-wall-related processes. Exponentially growing wild-type and deletion strains were pinned on solid YES medium containing 0.5 µg/ml micafungin and incubated at 30° for 3 days. (D) Cell-wall degradation assays. The Δomh1 strain was more sensitive to zymolyase treatment than wild type ( P = 1.6e −2 ). The zymolyase-resistant phenotype from overexpression of prz1 + was abrogated by loss of omh1 + ( P = 5.0e −4 ). The zymolyase experiments were repeated in triplicate, and error bars represent the standard error. The P -values were determined with ANOVA followed by a two-tailed t -test after 2 hr of zymolyase treatment.

    Article Snippet: Three milliliters of cell suspension was transferred to test tubes in the presence and absence of 25 U/ml Zymolyase 100T (Zymo Research) and shaken at 37°.

    Techniques: Expressing, Chromatin Immunoprecipitation, Microarray, Plasmid Preparation, Over Expression, Incubation, Two Tailed Test

    [ 14 C]Lysine cross-linked by TGase activity was found in low- and high-molecular-weight cell wall proteins and inhibition of TGase enzyme by cystamine and MDC induced changes in cell wall that sensitized cells to zymolyase treatment. A , paper chromatography of [ 14 C]putrescine-labeled material performed by C. albicans TGase activity and released from CW by zymolyase ( filled circles ). Solubilized fraction containing 20,000 cpm analyzed by paper chromatography was described under the “Experimental procedures.” [ 14 C]Putrescine substrate was also analyzed as a control ( open circles ). B , transglutaminase-mediated incorporation of [ 14 C]lysine into endogenous cell wall proteins. Reaction mixtures containing similar aliquots of cell walls and 2.5 μCi of [ 14 C]lysine with no cystamine ( open circles ) or with 50 m m cystamine ( filled circles ), boiled enzymatic source ( open triangles ), or 2 m m EDTA ( filled triangles ) were incubated for the indicated times; radioactivity in TCA-precipitable material was quantified. C , labeled samples with [ 14 C]lysine were sequentially released from cell walls by SDS, zymolyase, and chitinase and analyzed by 10% SDS-PAGE and fluorography. 10,000 cpm of labeled fractions were loaded in each lane. Lane 1, SDS-released material; lane 2 , zymolyase-released material; lane 3 , chitinase-released material. D and E, inhibition of TGase by cystamine and MDC increased sensitivity of C. albicans cells to treatment with zymolyase. Cells of C. albicans (adjusted to OD 600 nm = 0.5) previously incubated without ( circles ) or with 100 m m cystamine ( D ) or 3.5 m m MDC ( E ) for 1.5 h ( triangles ), 2 h ( inverted triangles ), and 4 h ( squares ) were treated with 50 μg ml −1 zymolyase 20T for up to 120 min. At the indicated times, OD 600 nm of cultures was monitored.

    Journal: The Journal of Biological Chemistry

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

    doi: 10.1074/jbc.M117.810440

    Figure Lengend Snippet: [ 14 C]Lysine cross-linked by TGase activity was found in low- and high-molecular-weight cell wall proteins and inhibition of TGase enzyme by cystamine and MDC induced changes in cell wall that sensitized cells to zymolyase treatment. A , paper chromatography of [ 14 C]putrescine-labeled material performed by C. albicans TGase activity and released from CW by zymolyase ( filled circles ). Solubilized fraction containing 20,000 cpm analyzed by paper chromatography was described under the “Experimental procedures.” [ 14 C]Putrescine substrate was also analyzed as a control ( open circles ). B , transglutaminase-mediated incorporation of [ 14 C]lysine into endogenous cell wall proteins. Reaction mixtures containing similar aliquots of cell walls and 2.5 μCi of [ 14 C]lysine with no cystamine ( open circles ) or with 50 m m cystamine ( filled circles ), boiled enzymatic source ( open triangles ), or 2 m m EDTA ( filled triangles ) were incubated for the indicated times; radioactivity in TCA-precipitable material was quantified. C , labeled samples with [ 14 C]lysine were sequentially released from cell walls by SDS, zymolyase, and chitinase and analyzed by 10% SDS-PAGE and fluorography. 10,000 cpm of labeled fractions were loaded in each lane. Lane 1, SDS-released material; lane 2 , zymolyase-released material; lane 3 , chitinase-released material. D and E, inhibition of TGase by cystamine and MDC increased sensitivity of C. albicans cells to treatment with zymolyase. Cells of C. albicans (adjusted to OD 600 nm = 0.5) previously incubated without ( circles ) or with 100 m m cystamine ( D ) or 3.5 m m MDC ( E ) for 1.5 h ( triangles ), 2 h ( inverted triangles ), and 4 h ( squares ) were treated with 50 μg ml −1 zymolyase 20T for up to 120 min. At the indicated times, OD 600 nm of cultures was monitored.

    Article Snippet: Identification of TGase protein using a fluorescent TGase inhibitor MDC probe Cell walls from C. albicans were simultaneously digested with 60 units of chitinase (Sigma) and 100 units of zymolyase (Zymo Research) overnight at room temperature.

    Techniques: Activity Assay, Molecular Weight, Inhibition, Paper Chromatography, Labeling, Incubation, Radioactivity, SDS Page

    Active Cdc42 interferes with cytokinesis and cell separation. (A) Cells transformed with vector (VEC) or GAL1-HA-CDC42 plasmids were spotted in fivefold dilutions on the indicated media (3 d at 25°C). OE, overexpression; GAL, galactose. (B) Expression of cdc42 G60D from the CDC42 promoter on a CEN plasmid (one to three copies per cell) is toxic to myo1Δ cells. Cells were grown on the indicated media (3 d at 25°C). Synthetic lethality is detected on 5-fluoroorotic acid (5FOA) media as a counterselection for the indicated URA3 -containing plasmids. (C) myo1Δ cells transformed with control or CDC24 2μ plasmids were grown on plates as in B. SC, synthetic complete. (D) Wild-type (wt) and bem2-ts cells were arrested in metaphase by CDC20 depletion, shifted to the restrictive temperature (37°C at 1.5 h), and processed for Cdc42 activation. Graph shows means ± SEM from three experiments. (E) Cells were arrested in metaphase, shifted to 37°C for 1 h, and released at 37°C. The percentage of cells with the indicated morphology is plotted as means ± SEM from three experiments. Connected indicates two adjacent cell bodies showing evidence of rebudding or repolarization. (F) Bright-field image of cells 50 min after release. Bar, 15 µm. (G) Cells from E were treated with zymolyase to digest the cell wall, and the percentage of large-budded or connected cells was scored. (H) Wild-type cells have a PS (arrow) sandwiched by secondary septa; bem2-ts cells have misaligned, multiple, or otherwise abnormal PSs (arrowheads). Bars, 500 nm.

    Journal: The Journal of Cell Biology

    Article Title: Inhibition of Cdc42 during mitotic exit is required for cytokinesis

    doi: 10.1083/jcb.201301090

    Figure Lengend Snippet: Active Cdc42 interferes with cytokinesis and cell separation. (A) Cells transformed with vector (VEC) or GAL1-HA-CDC42 plasmids were spotted in fivefold dilutions on the indicated media (3 d at 25°C). OE, overexpression; GAL, galactose. (B) Expression of cdc42 G60D from the CDC42 promoter on a CEN plasmid (one to three copies per cell) is toxic to myo1Δ cells. Cells were grown on the indicated media (3 d at 25°C). Synthetic lethality is detected on 5-fluoroorotic acid (5FOA) media as a counterselection for the indicated URA3 -containing plasmids. (C) myo1Δ cells transformed with control or CDC24 2μ plasmids were grown on plates as in B. SC, synthetic complete. (D) Wild-type (wt) and bem2-ts cells were arrested in metaphase by CDC20 depletion, shifted to the restrictive temperature (37°C at 1.5 h), and processed for Cdc42 activation. Graph shows means ± SEM from three experiments. (E) Cells were arrested in metaphase, shifted to 37°C for 1 h, and released at 37°C. The percentage of cells with the indicated morphology is plotted as means ± SEM from three experiments. Connected indicates two adjacent cell bodies showing evidence of rebudding or repolarization. (F) Bright-field image of cells 50 min after release. Bar, 15 µm. (G) Cells from E were treated with zymolyase to digest the cell wall, and the percentage of large-budded or connected cells was scored. (H) Wild-type cells have a PS (arrow) sandwiched by secondary septa; bem2-ts cells have misaligned, multiple, or otherwise abnormal PSs (arrowheads). Bars, 500 nm.

    Article Snippet: In brief, for zymolyase treatment, cells were fixed for 1 h with 3.7% formaldehyde, washed three times with PBS and twice with KS buffer (1.2 M sorbitol and 50 mM KHPO4 ), and then resuspended in KS containing 0.2 mg/ml zymolyase (Zymo Research) and 2 mM DTT until > 90% of cells lost their refractive appearance.

    Techniques: Transformation Assay, Plasmid Preparation, Over Expression, Expressing, Activation Assay

    Ag displayed on the surface of yeast is cross-presented more efficiently than Ag expressed intracellularly in yeast. A , Diagram of the yeast surface display system of strain EBYN9V. The N9V epitope is in bold. B , Dose response of EBYN9V on cross-presentation. EBYN9V and wild-type EBY100 yeast were added to DCs at the indicated ratios. After 24 h, the DCs were assayed for the ability to stimulate IFN- γ secretion in cocultured N9V-specific T cells. Error bars, SDs of duplicate wells. C , EBYN9V yeast and yeast expressing the same Aga2p-N9V fusion protein intracellularly were added to DCs at a 20:1 ratio and tested for the ability to stimulate IFN- γ secretion in cocultured N9V-specific T cells. Lactacystin (5 μ M) or chloroquine (25 μ M) were added to some wells an hour before the yeast were introduced. Error bars, SDs of duplicate wells. D , Samples of the two yeast cultures and wt yeast were subjected to extensive reduction to release proteins disulfide-bonded to the cell wall. Subsequently, the yeast were treated with Zymolyase and lysed. The proteins reduced off the cell wall and the lysed cell extracts were slot-blotted onto the same nitrocellulose membrane and labeled for c- myc .

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Antigen Release Kinetics in the Phagosome Are Critical to Cross-Presentation Efficiency

    doi:

    Figure Lengend Snippet: Ag displayed on the surface of yeast is cross-presented more efficiently than Ag expressed intracellularly in yeast. A , Diagram of the yeast surface display system of strain EBYN9V. The N9V epitope is in bold. B , Dose response of EBYN9V on cross-presentation. EBYN9V and wild-type EBY100 yeast were added to DCs at the indicated ratios. After 24 h, the DCs were assayed for the ability to stimulate IFN- γ secretion in cocultured N9V-specific T cells. Error bars, SDs of duplicate wells. C , EBYN9V yeast and yeast expressing the same Aga2p-N9V fusion protein intracellularly were added to DCs at a 20:1 ratio and tested for the ability to stimulate IFN- γ secretion in cocultured N9V-specific T cells. Lactacystin (5 μ M) or chloroquine (25 μ M) were added to some wells an hour before the yeast were introduced. Error bars, SDs of duplicate wells. D , Samples of the two yeast cultures and wt yeast were subjected to extensive reduction to release proteins disulfide-bonded to the cell wall. Subsequently, the yeast were treated with Zymolyase and lysed. The proteins reduced off the cell wall and the lysed cell extracts were slot-blotted onto the same nitrocellulose membrane and labeled for c- myc .

    Article Snippet: The yeast pellets were then washed with spheroplast buffer (50 mM Tris-HCl, pH 7.5, 1.4 M sorbitol, and 40 mM 2-ME), incubated with 2.4 U Zymolyase (Zymo Research) in 120 μ l spheroplast buffer containing a protease inhibitor mixture (Roche) for 15 min at 37°C, and boiled in 2% SDS for 5 min.

    Techniques: Expressing, Labeling