β lactamase  (Millipore)


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    Name:
    beta Lactamase
    Description:
    β Lactamase produced by bacteria is closely related to the penicillin binding proteins β Lactamase hydrolyzes β lactum antibiotics and is the prime cause of resistance development by bacteria There are four subclasses of β Lactamases Classes A C and D form an acyl enzyme via active site serine residue Class B β lactamases are metalloenzymes with zinc ion at their active site for β lactam hydrolysis Mutations in the β lactamases has resulted in the generation of extended spectrum β lactamases ESBLs As close to 900 types of β Lactamases are produced by microbes
    Catalog Number:
    l6170
    Price:
    None
    Applications:
    β--lactamase is used to inactivate β-lactam antibiotics by breaking open the β-lactam ring. β--lactamase is used to study antibiotic resistance and resistance suppression. Product L6170 is recombinantly produced based on the sequence of the enzyme from Pseudomonas aeruginosa, and is expressed in E. coli.
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    Structured Review

    Millipore β lactamase
    Method for synthetic evolution of the translation initiation region (TIR). a Schematic of the pET28a-His 6 -TPS-sfGFP-translational coupling <t>device-β-lactamase</t> expression cassette. The plasmid encodes for a His 6 -tag, a thrombin protease site (TPS), sfGFP (green arrow), followed by a translational coupling device (hp; weak coupling 1) and the reporter, β-lactamase (bla; purple arrow). The boxed area represents the TIR for the expression cassette. b The pET28a-His 6 -sfGFP-hp-bla plasmid was used as a template for the creation of TIR LIBRARIES ; degenerate primers used to create a TIR LIBRARY with synonymous codon changes and one with non-synonymous codon changes. c Following preparation of a TIR LIBRARY plasmids were transformed into E. coli BL21( DE3 ) pLysS. TIR LIBRARY variants that showed higher levels of protein expression are able to grow on plates at higher concentrations of ampicillin relative to the standard pET28a and were selected.
    β Lactamase produced by bacteria is closely related to the penicillin binding proteins β Lactamase hydrolyzes β lactum antibiotics and is the prime cause of resistance development by bacteria There are four subclasses of β Lactamases Classes A C and D form an acyl enzyme via active site serine residue Class B β lactamases are metalloenzymes with zinc ion at their active site for β lactam hydrolysis Mutations in the β lactamases has resulted in the generation of extended spectrum β lactamases ESBLs As close to 900 types of β Lactamases are produced by microbes
    https://www.bioz.com/result/β lactamase/product/Millipore
    Average 95 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    β lactamase - by Bioz Stars, 2020-07
    95/100 stars

    Images

    1) Product Images from "Improved designs for pET expression plasmids increase protein production yield in Escherichia coli"

    Article Title: Improved designs for pET expression plasmids increase protein production yield in Escherichia coli

    Journal: Communications Biology

    doi: 10.1038/s42003-020-0939-8

    Method for synthetic evolution of the translation initiation region (TIR). a Schematic of the pET28a-His 6 -TPS-sfGFP-translational coupling device-β-lactamase expression cassette. The plasmid encodes for a His 6 -tag, a thrombin protease site (TPS), sfGFP (green arrow), followed by a translational coupling device (hp; weak coupling 1) and the reporter, β-lactamase (bla; purple arrow). The boxed area represents the TIR for the expression cassette. b The pET28a-His 6 -sfGFP-hp-bla plasmid was used as a template for the creation of TIR LIBRARIES ; degenerate primers used to create a TIR LIBRARY with synonymous codon changes and one with non-synonymous codon changes. c Following preparation of a TIR LIBRARY plasmids were transformed into E. coli BL21( DE3 ) pLysS. TIR LIBRARY variants that showed higher levels of protein expression are able to grow on plates at higher concentrations of ampicillin relative to the standard pET28a and were selected.
    Figure Legend Snippet: Method for synthetic evolution of the translation initiation region (TIR). a Schematic of the pET28a-His 6 -TPS-sfGFP-translational coupling device-β-lactamase expression cassette. The plasmid encodes for a His 6 -tag, a thrombin protease site (TPS), sfGFP (green arrow), followed by a translational coupling device (hp; weak coupling 1) and the reporter, β-lactamase (bla; purple arrow). The boxed area represents the TIR for the expression cassette. b The pET28a-His 6 -sfGFP-hp-bla plasmid was used as a template for the creation of TIR LIBRARIES ; degenerate primers used to create a TIR LIBRARY with synonymous codon changes and one with non-synonymous codon changes. c Following preparation of a TIR LIBRARY plasmids were transformed into E. coli BL21( DE3 ) pLysS. TIR LIBRARY variants that showed higher levels of protein expression are able to grow on plates at higher concentrations of ampicillin relative to the standard pET28a and were selected.

    Techniques Used: Expressing, Plasmid Preparation, Transformation Assay

    2) Product Images from "Combinatorial microfluidic droplet engineering for biomimetic material synthesis"

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis

    Journal: Science Advances

    doi: 10.1126/sciadv.1600567

    Mineral shells formed from [W/O/W] double emulsions support in vitro protein expression. ( A ) Schematic overview of the second, double emulsion–producing, flow-focusing junction of a two-junction flow-focusing microfluidic device used to generate mineralized double emulsions (see fig. S10 and movie S4). ( B ) Schematic of [W/O/W] droplet mineralization to form an interfacial mineral layer around bacterial extracts in the innermost water phase. Here, “S” is a fluorogenic substrate (CCF2) that changes emission properties when enzymatically converted to product (“P”) by β-lactamase (β-lac) enzyme. ( C ) Polarized light optical micrograph shows interfacial birefringence. ( D ) Flow cytometry scatter plots of 50,000 droplets, proving that mineralized droplets support compartmentalized in vitro protein expression.
    Figure Legend Snippet: Mineral shells formed from [W/O/W] double emulsions support in vitro protein expression. ( A ) Schematic overview of the second, double emulsion–producing, flow-focusing junction of a two-junction flow-focusing microfluidic device used to generate mineralized double emulsions (see fig. S10 and movie S4). ( B ) Schematic of [W/O/W] droplet mineralization to form an interfacial mineral layer around bacterial extracts in the innermost water phase. Here, “S” is a fluorogenic substrate (CCF2) that changes emission properties when enzymatically converted to product (“P”) by β-lactamase (β-lac) enzyme. ( C ) Polarized light optical micrograph shows interfacial birefringence. ( D ) Flow cytometry scatter plots of 50,000 droplets, proving that mineralized droplets support compartmentalized in vitro protein expression.

    Techniques Used: In Vitro, Expressing, Flow Cytometry, Cytometry

    3) Product Images from "Non-canonical autophagy in dendritic cells restricts cross-presentation and anti-tumor immunity"

    Article Title: Non-canonical autophagy in dendritic cells restricts cross-presentation and anti-tumor immunity

    Journal: bioRxiv

    doi: 10.1101/789867

    Rubcn -/- DCs exhibit increased phagosome-to-cytosol escape and proteasome-mediated generation of peptides. Bone marrow-derived dendritic cells (DCs) were generated from Rubcn +/+ (black) and Rubcn -/- (red) mice in vitro with FLT3-L for 7 days. ( A-B ) DCs were loaded with 1 µm CCF4, then co-cultured with apoptotic B16-OVA in the absence or presence of β-lactamase (2 mg/ml) for 90 or 180 minutes at 4°C or 37°C. Uncleaved CCF4 was measured by flow cytometry at an emission of 535 nm, and cleaved CCF4 was measured at an emission of 450 nm. Ratios of 450 nm : 535 nm was calculated by dividing the 450 nm MFI by the 535 nm MFI from a single sample. ( C ) DCs were pre-treated with vehicle or chloroquine (CQ) at 1 or 10 µM for 2 hours and then co-cultured with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. ( D ) DCs were pre-treated with vehicle or MG-132 at 1 or 10 µM for 2 hours and then co-cultured in fresh media with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. ( E ) DCs were pre-treated with vehicle or Brefeldin A at 3 µg/ml for 2 hours and then co-cultured in fresh media with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. Data are expressed as mean ± SEM. No less than two independent experiments were performed, with 3-5 replicates per condition. Significance was calculated using 2-way ANOVA (*p
    Figure Legend Snippet: Rubcn -/- DCs exhibit increased phagosome-to-cytosol escape and proteasome-mediated generation of peptides. Bone marrow-derived dendritic cells (DCs) were generated from Rubcn +/+ (black) and Rubcn -/- (red) mice in vitro with FLT3-L for 7 days. ( A-B ) DCs were loaded with 1 µm CCF4, then co-cultured with apoptotic B16-OVA in the absence or presence of β-lactamase (2 mg/ml) for 90 or 180 minutes at 4°C or 37°C. Uncleaved CCF4 was measured by flow cytometry at an emission of 535 nm, and cleaved CCF4 was measured at an emission of 450 nm. Ratios of 450 nm : 535 nm was calculated by dividing the 450 nm MFI by the 535 nm MFI from a single sample. ( C ) DCs were pre-treated with vehicle or chloroquine (CQ) at 1 or 10 µM for 2 hours and then co-cultured with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. ( D ) DCs were pre-treated with vehicle or MG-132 at 1 or 10 µM for 2 hours and then co-cultured in fresh media with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. ( E ) DCs were pre-treated with vehicle or Brefeldin A at 3 µg/ml for 2 hours and then co-cultured in fresh media with apoptotic B16-OVA cells (5 apoptotic cells: 1 DC). Eighteen hours later, DCs were harvested for flow cytometry analysis of H2-K b -OVA 257-264 expression. Data are expressed as mean ± SEM. No less than two independent experiments were performed, with 3-5 replicates per condition. Significance was calculated using 2-way ANOVA (*p

    Techniques Used: Derivative Assay, Generated, Mouse Assay, In Vitro, Cell Culture, Flow Cytometry, Expressing

    4) Product Images from "Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway"

    Article Title: Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04985-0

    Human mo-DCs and mo-Mac are inefficient for the transfer of exogenous proteins into their cytosol. a , b Purified DCs and macrophages from tumor ascites, derived in vitro from monocytes, or DCs derived in vitro from CD34 + precursors were loaded with a cell-permeable FRET-sensitive substrate of β-lactamase, and incubated with or without exogenous β-lactamase. After 3 h, cleavage was measured by flow cytometry. a Representative results of six (tumor ascites), ten (in vitro monocyte-derived), or eight (in vitro CD34 + cell-derived) independent experiments. b Quantification of β-lactamase transfer. Symbols represent individual donors. N = 6 for tumor ascites, N = 10 for in vitro monocyte-derived cells, and N = 8 for CD34 + cell-derived cells. ** p
    Figure Legend Snippet: Human mo-DCs and mo-Mac are inefficient for the transfer of exogenous proteins into their cytosol. a , b Purified DCs and macrophages from tumor ascites, derived in vitro from monocytes, or DCs derived in vitro from CD34 + precursors were loaded with a cell-permeable FRET-sensitive substrate of β-lactamase, and incubated with or without exogenous β-lactamase. After 3 h, cleavage was measured by flow cytometry. a Representative results of six (tumor ascites), ten (in vitro monocyte-derived), or eight (in vitro CD34 + cell-derived) independent experiments. b Quantification of β-lactamase transfer. Symbols represent individual donors. N = 6 for tumor ascites, N = 10 for in vitro monocyte-derived cells, and N = 8 for CD34 + cell-derived cells. ** p

    Techniques Used: Purification, Derivative Assay, In Vitro, Incubation, Flow Cytometry, Cytometry

    5) Product Images from "Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing"

    Article Title: Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0700970104

    The R18-dequenching lipid mixing assay. ( A ) Vacuoles were labeled with R18, mixed with unlabeled vacuoles in lipid mixing assay reactions containing anti-Sec17p (154 nM), anti-Sec18p (142 nM), anti-Ypt7p (167 nM), Gdi1p (1.2 μM)/Gyp1–46p (5 μM), anti-Vam3p (444 nM), anti-Nyv1p (261 nM), MED (10 μM), 3sQ-SNARE (2 μM), recombinant Sec18p (63 nM), rVam7p (40 nM), or buffer only (No addition), and lipid mixing was monitored at 27°C. One reaction did not receive unlabeled acceptor vacuoles (open circles). ( B ) After 90 min, ALP activity was measured. ( C and D ) Vacuoles isolated from BJ3505-Fos-ω were labeled with R18, reisolated, and added to lipid mixing assay reactions containing unlabeled DKY6281 vacuoles ( C ) or unlabeled vacuoles from BJ3505-α-Jun ( D ). Lipid mixing (squares) was monitored by R18 dequenching under low Mg 2+ condition at 27°C for 90 min with the indicated concentrations of GTPγS. After 90 min, the reactions were assayed for ALP activity ( C ; circles) or β-lactamase activity ( D ; circles). Values obtained without inhibitors [2.31 ± 0.46 units for the ALP assay coupled with the R18 assay (Δ F 90min / F TX100 = 0.102 ± 0.047); 1.53 ± 0.07 units for the β-lactamase assay coupled with the R18 assay (Δ F 90min / F TX100 = 0.201 ± 0.023)] were used as the 100% reference. Data represent mean ± SEM ( n = 3).
    Figure Legend Snippet: The R18-dequenching lipid mixing assay. ( A ) Vacuoles were labeled with R18, mixed with unlabeled vacuoles in lipid mixing assay reactions containing anti-Sec17p (154 nM), anti-Sec18p (142 nM), anti-Ypt7p (167 nM), Gdi1p (1.2 μM)/Gyp1–46p (5 μM), anti-Vam3p (444 nM), anti-Nyv1p (261 nM), MED (10 μM), 3sQ-SNARE (2 μM), recombinant Sec18p (63 nM), rVam7p (40 nM), or buffer only (No addition), and lipid mixing was monitored at 27°C. One reaction did not receive unlabeled acceptor vacuoles (open circles). ( B ) After 90 min, ALP activity was measured. ( C and D ) Vacuoles isolated from BJ3505-Fos-ω were labeled with R18, reisolated, and added to lipid mixing assay reactions containing unlabeled DKY6281 vacuoles ( C ) or unlabeled vacuoles from BJ3505-α-Jun ( D ). Lipid mixing (squares) was monitored by R18 dequenching under low Mg 2+ condition at 27°C for 90 min with the indicated concentrations of GTPγS. After 90 min, the reactions were assayed for ALP activity ( C ; circles) or β-lactamase activity ( D ; circles). Values obtained without inhibitors [2.31 ± 0.46 units for the ALP assay coupled with the R18 assay (Δ F 90min / F TX100 = 0.102 ± 0.047); 1.53 ± 0.07 units for the β-lactamase assay coupled with the R18 assay (Δ F 90min / F TX100 = 0.201 ± 0.023)] were used as the 100% reference. Data represent mean ± SEM ( n = 3).

    Techniques Used: Labeling, Recombinant, ALP Assay, Activity Assay, Isolation, Beta Lactamase Assay

    6) Product Images from "The Avr (Effector) Proteins HrmA (HopPsyA) and AvrPto Are Secreted in Culture from Pseudomonas syringae Pathovars via the Hrp (Type III) Protein Secretion System in a Temperature- and pH-Sensitive Manner"

    Article Title: The Avr (Effector) Proteins HrmA (HopPsyA) and AvrPto Are Secreted in Culture from Pseudomonas syringae Pathovars via the Hrp (Type III) Protein Secretion System in a Temperature- and pH-Sensitive Manner

    Journal: Journal of Bacteriology

    doi:

    Native AvrB cannot be detected in culture supernatants from P. syringae pv. glycinea race 0, even though heterologously expressed AvrPto is secreted. (A) P. syringae pv. glycinea ( Psg ) race 0(pCPP2318) was grown at 20°C in hrp -derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. The fractions were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting using either anti-AvrPto or anti-β-lactamase antibodies. (B) P. syringae pv. glycinea race 0(pCPP2318, pCPP3026) was grown at 20°C in hrp -derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. pCPP3026 encodes AvrPto from P. syringae pv. tomato DC3000. After separation of proteins by SDS-PAGE, immunoblot analysis was carried out with anti-AvrB, anti-AvrPto, or anti-β-lactamase antibodies. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
    Figure Legend Snippet: Native AvrB cannot be detected in culture supernatants from P. syringae pv. glycinea race 0, even though heterologously expressed AvrPto is secreted. (A) P. syringae pv. glycinea ( Psg ) race 0(pCPP2318) was grown at 20°C in hrp -derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. The fractions were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting using either anti-AvrPto or anti-β-lactamase antibodies. (B) P. syringae pv. glycinea race 0(pCPP2318, pCPP3026) was grown at 20°C in hrp -derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. pCPP3026 encodes AvrPto from P. syringae pv. tomato DC3000. After separation of proteins by SDS-PAGE, immunoblot analysis was carried out with anti-AvrB, anti-AvrPto, or anti-β-lactamase antibodies. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.

    Techniques Used: SDS Page, Software

    Distribution of HrmA and β-lactamase in cultures of P. syringae pv. syringae 61(pCPP2318) or hrp mutant P. syringae pv. syringae 61-2089(pCPP2318). Bacterial cultures were grown at 25°C in hrp -derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. The cell-bound fractions were concentrated 13.4-fold and the supernatant fractions were concentrated 100-fold relative to the initial culture volumes. The samples were subjected to SDS-PAGE and immunoblot analysis, and HrmA and β-lactamase were detected with either anti-HrmA or anti-β-lactamase antibodies followed by secondary antibodies conjugated to alkaline phosphatase as described in Materials and Methods. Pss wild-type, P. syringae pv. syringae 61(pCPP2318); Pss hrcC , P. syringae pv. syringae 61-2089(pCPP2318). The image of the immunoblot was captured using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
    Figure Legend Snippet: Distribution of HrmA and β-lactamase in cultures of P. syringae pv. syringae 61(pCPP2318) or hrp mutant P. syringae pv. syringae 61-2089(pCPP2318). Bacterial cultures were grown at 25°C in hrp -derepressing medium and separated into cell-bound (C) and supernatant (S) fractions. The cell-bound fractions were concentrated 13.4-fold and the supernatant fractions were concentrated 100-fold relative to the initial culture volumes. The samples were subjected to SDS-PAGE and immunoblot analysis, and HrmA and β-lactamase were detected with either anti-HrmA or anti-β-lactamase antibodies followed by secondary antibodies conjugated to alkaline phosphatase as described in Materials and Methods. Pss wild-type, P. syringae pv. syringae 61(pCPP2318); Pss hrcC , P. syringae pv. syringae 61-2089(pCPP2318). The image of the immunoblot was captured using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.

    Techniques Used: Mutagenesis, SDS Page, Software

    The secretion in culture of HrmA (HopPsyA) is affected by temperature, whereas the secretion of HrpZ is not. P. syringae pv. syringae 61(pCPP2318) cultures were grown in hrp -derepressing medium at the temperatures indicated. Bacterial cultures were separated into cell-bound (C) and supernatant (S) fractions by centrifugation, and the supernatant fractions were adjusted to be 7.5 times more concentrated than the cell-bound fractions. After the samples were separated by SDS-PAGE and transferred to PVDF membranes, HrmA, HrpZ, and β-lactamase were detected by immunoblotting using anti-HrmA, anti-HrpZ, and anti-β-lactamase antibodies, respectively, followed by secondary antibodies conjugated to alkaline phosphatase. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
    Figure Legend Snippet: The secretion in culture of HrmA (HopPsyA) is affected by temperature, whereas the secretion of HrpZ is not. P. syringae pv. syringae 61(pCPP2318) cultures were grown in hrp -derepressing medium at the temperatures indicated. Bacterial cultures were separated into cell-bound (C) and supernatant (S) fractions by centrifugation, and the supernatant fractions were adjusted to be 7.5 times more concentrated than the cell-bound fractions. After the samples were separated by SDS-PAGE and transferred to PVDF membranes, HrmA, HrpZ, and β-lactamase were detected by immunoblotting using anti-HrmA, anti-HrpZ, and anti-β-lactamase antibodies, respectively, followed by secondary antibodies conjugated to alkaline phosphatase. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.

    Techniques Used: Centrifugation, SDS Page, Software

    The pH of the growth medium affects the secretion in culture of HrmA (HopPsyA) and HrpZ via the Hrp secretion system. P. syringae pv. syringae 61(pCPP2318) cultures were grown at 22°C in hrp -derepressing media differing only in the pH of the medium. Bacterial cultures were separated into cell-bound (C) and supernatant (S) fractions by centrifugation, and proteins were separated by SDS-PAGE. Immunoblot analysis was carried out as described in Materials and Methods, using anti-HrmA, anti-HrpZ, or anti-β-lactamase antibodies followed by secondary antibodies conjugated to alkaline phosphatase. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
    Figure Legend Snippet: The pH of the growth medium affects the secretion in culture of HrmA (HopPsyA) and HrpZ via the Hrp secretion system. P. syringae pv. syringae 61(pCPP2318) cultures were grown at 22°C in hrp -derepressing media differing only in the pH of the medium. Bacterial cultures were separated into cell-bound (C) and supernatant (S) fractions by centrifugation, and proteins were separated by SDS-PAGE. Immunoblot analysis was carried out as described in Materials and Methods, using anti-HrmA, anti-HrpZ, or anti-β-lactamase antibodies followed by secondary antibodies conjugated to alkaline phosphatase. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.

    Techniques Used: Centrifugation, SDS Page, Software

    AvrPto is secreted in culture from P. syringae pv. tomato DC3000 via the Hrp secretion system at 20°C but not at 30°C. P. syringae pv. tomato DC3000(pCPP2318) cultures were grown in hrp -derepressing medium at 20 and 30°C. The supernatant (S) and cell-bound (C) fractions were isolated as before, separated by SDS-PAGE, and analyzed by immunoblotting with anti-AvrPto or anti-β-lactamase antibodies. The preparations of the protein samples resulted in supernatant fractions that were concentrated 7.5 times more than the cell-bound fractions. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
    Figure Legend Snippet: AvrPto is secreted in culture from P. syringae pv. tomato DC3000 via the Hrp secretion system at 20°C but not at 30°C. P. syringae pv. tomato DC3000(pCPP2318) cultures were grown in hrp -derepressing medium at 20 and 30°C. The supernatant (S) and cell-bound (C) fractions were isolated as before, separated by SDS-PAGE, and analyzed by immunoblotting with anti-AvrPto or anti-β-lactamase antibodies. The preparations of the protein samples resulted in supernatant fractions that were concentrated 7.5 times more than the cell-bound fractions. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.

    Techniques Used: Isolation, SDS Page, Software

    AvrPto is secreted in culture from P. syringae pv. tomato DC3000 at pH 6.0 but not at pH 7.0. P. syringae pv. tomato DC3000(pCPP2318) cultures were grown at 20°C in hrp -derepressing medium adjusted to either pH 6.0 or pH 7.0. Isolated cell-bound (C) and supernatant (S) fractions were separated by SDS-PAGE and analyzed by immunoblotting using anti-AvrPto or anti-β-lactamase antibodies. Mature β-lactamase encoded by pCPP2318 was included as a control for cell lysis. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.
    Figure Legend Snippet: AvrPto is secreted in culture from P. syringae pv. tomato DC3000 at pH 6.0 but not at pH 7.0. P. syringae pv. tomato DC3000(pCPP2318) cultures were grown at 20°C in hrp -derepressing medium adjusted to either pH 6.0 or pH 7.0. Isolated cell-bound (C) and supernatant (S) fractions were separated by SDS-PAGE and analyzed by immunoblotting using anti-AvrPto or anti-β-lactamase antibodies. Mature β-lactamase encoded by pCPP2318 was included as a control for cell lysis. The image of the immunoblot was captured by using the Bio-Rad Gel Doc 1000 UV fluorescent gel documentation system with the accompanying Multi-Analyst PC software. For figure construction, the image was manipulated by using Microsoft PowerPoint 97 and transferred to Adobe Photoshop 4.0 to meet the publisher’s specifications.

    Techniques Used: Isolation, SDS Page, Lysis, Software

    7) Product Images from "Lipid peroxidation causes endosomal antigen release for cross-presentation"

    Article Title: Lipid peroxidation causes endosomal antigen release for cross-presentation

    Journal: Scientific Reports

    doi: 10.1038/srep22064

    Lipid peroxidation causes antigen release from endosomes. ( a ) Liposomes with lysozyme encapsulated were treated with a combination of H 2 O 2 and Fe(II)SO 4 (ROS) or Triton X-100 (TX100; positive control) followed by incubation with trypsin. Samples were analyzed by SDS-PAGE. Graph shows semi-quantifications of band intensities from 4 independent experiments (mean ± S.E.M). ( b ) Scheme and confocal microscope images of a CCF4 assay. The cytosolic FRET probe CCF4 was cleaved by exogenous β-lactamase (β-lac) resulting in a decreased ratio of fluorescein (acceptor fluorophore; green) over coumarin (donor; blue) fluorescence. BF: bright field. ( c–d ) CCF4 cleavage efficiencies for DCs cultured in the absence or presence of LPS or α-tocopherol (α-Toc) ( c ) and for NOX2 KD DCs ( d ). NT: non-targeting siRNA. ( e–f ) Cell viability by the MTT assay of cells cultured with exogenous cytochrome C (Cyt C) and in presence or absence of α-tocopherol (α-Toc) ( e ) as well as for NOX2 KD DCs ( f ). Each donor is plotted in a different color. ( g ) Confocal micrographs of an unstimulated (no LPS) and stimulated (+LPS) DC expressing galectin-3-mAzami (green), incubated with OVA conjugated to Alexa fluor 647 (OVA-AF647; red). ( h ) Fluorescence intensities of galectin-3-mAzami thresholded on OVA-AF647, relative to cytosolic fluorescence intensity, in DCs in the presence or absence of LPS and α-Toc. Results show individual results for at least 3 donors. Scale bars, 10 μm.
    Figure Legend Snippet: Lipid peroxidation causes antigen release from endosomes. ( a ) Liposomes with lysozyme encapsulated were treated with a combination of H 2 O 2 and Fe(II)SO 4 (ROS) or Triton X-100 (TX100; positive control) followed by incubation with trypsin. Samples were analyzed by SDS-PAGE. Graph shows semi-quantifications of band intensities from 4 independent experiments (mean ± S.E.M). ( b ) Scheme and confocal microscope images of a CCF4 assay. The cytosolic FRET probe CCF4 was cleaved by exogenous β-lactamase (β-lac) resulting in a decreased ratio of fluorescein (acceptor fluorophore; green) over coumarin (donor; blue) fluorescence. BF: bright field. ( c–d ) CCF4 cleavage efficiencies for DCs cultured in the absence or presence of LPS or α-tocopherol (α-Toc) ( c ) and for NOX2 KD DCs ( d ). NT: non-targeting siRNA. ( e–f ) Cell viability by the MTT assay of cells cultured with exogenous cytochrome C (Cyt C) and in presence or absence of α-tocopherol (α-Toc) ( e ) as well as for NOX2 KD DCs ( f ). Each donor is plotted in a different color. ( g ) Confocal micrographs of an unstimulated (no LPS) and stimulated (+LPS) DC expressing galectin-3-mAzami (green), incubated with OVA conjugated to Alexa fluor 647 (OVA-AF647; red). ( h ) Fluorescence intensities of galectin-3-mAzami thresholded on OVA-AF647, relative to cytosolic fluorescence intensity, in DCs in the presence or absence of LPS and α-Toc. Results show individual results for at least 3 donors. Scale bars, 10 μm.

    Techniques Used: Positive Control, Incubation, SDS Page, Microscopy, Fluorescence, Cell Culture, MTT Assay, Expressing

    8) Product Images from "Role of Yersinia pestis Toxin Complex Family Proteins in Resistance to Phagocytosis by Polymorphonuclear Leukocytes"

    Article Title: Role of Yersinia pestis Toxin Complex Family Proteins in Resistance to Phagocytosis by Polymorphonuclear Leukocytes

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00648-13

    YopM– and YopE–β-lactamase are translocated into CHO cells and PMNs via the T3SS, but YitA– and YipA–β-lactamase are not. (A) Representative images of CHO cells and human PMNs that were loaded with the fluorescent β-lactamase substrate CCF2-AM and incubated with Y. pestis KIM5(pMM85), expressing YopE–β-lactamase, KIM5(pCR-XL-TOPO:: yitR ), expressing YitA–β-lactamase, or KIM5(pCR-XL-TOPO:: yitR ), expressing YipA–β-lactamase. Intact CCF2-AM fluoresces green, and β-lactamase-cleaved CCF2-AM fluoresces blue. The cumulative flow cytometry results for three separate experiments are shown as the percentages of CHO cells (B) and PMNs (C) that contained cleaved CCF2-AM following incubation with Y. pestis with (KIM5) or without (KIM6) the T3SS-encoding pCD1 and expressing either YitA–, YipA–, YopE–, or YopM–β-lactamase. Uninfected cells or cells incubated with KIM5 lacking β-lactamase fusion proteins were used as negative controls. The means and standard errors are indicated. Data were analyzed by one-way ANOVA with the Bonferroni posttest. *, P
    Figure Legend Snippet: YopM– and YopE–β-lactamase are translocated into CHO cells and PMNs via the T3SS, but YitA– and YipA–β-lactamase are not. (A) Representative images of CHO cells and human PMNs that were loaded with the fluorescent β-lactamase substrate CCF2-AM and incubated with Y. pestis KIM5(pMM85), expressing YopE–β-lactamase, KIM5(pCR-XL-TOPO:: yitR ), expressing YitA–β-lactamase, or KIM5(pCR-XL-TOPO:: yitR ), expressing YipA–β-lactamase. Intact CCF2-AM fluoresces green, and β-lactamase-cleaved CCF2-AM fluoresces blue. The cumulative flow cytometry results for three separate experiments are shown as the percentages of CHO cells (B) and PMNs (C) that contained cleaved CCF2-AM following incubation with Y. pestis with (KIM5) or without (KIM6) the T3SS-encoding pCD1 and expressing either YitA–, YipA–, YopE–, or YopM–β-lactamase. Uninfected cells or cells incubated with KIM5 lacking β-lactamase fusion proteins were used as negative controls. The means and standard errors are indicated. Data were analyzed by one-way ANOVA with the Bonferroni posttest. *, P

    Techniques Used: Incubation, Expressing, Flow Cytometry, Cytometry

    Detection of T3SS-dependent secretion of YopE– and YopM–β-lactamase but not YitA– or YipA–β-lactamase in culture supernatants. Y. pestis (pCR-XL-TOPO:: yitR ) strains with (KIM5) or without (KIM6) the pCD1 virulence plasmid encoding the T3SS and engineered to express YitA–, YipA–, YopM– (pMM83), or YopE–β-lactamase (pMM85) were grown in TMH overnight at 21°C, normalized, and then subcultured in TMH (without CaCl 2 ) and incubated at 21°C (gray columns) or 37°C (black columns) for an additional 4 h. Bacterial lysates (A) and culture supernatants (B) were incubated with fluorocillin for 4 h, and the fluorescence of each sample was measured. The means and standard errors for three experiments are indicated. Y. pestis KIM5 lacking β-lactamase fusion proteins and sterile TMH medium were used as background controls.
    Figure Legend Snippet: Detection of T3SS-dependent secretion of YopE– and YopM–β-lactamase but not YitA– or YipA–β-lactamase in culture supernatants. Y. pestis (pCR-XL-TOPO:: yitR ) strains with (KIM5) or without (KIM6) the pCD1 virulence plasmid encoding the T3SS and engineered to express YitA–, YipA–, YopM– (pMM83), or YopE–β-lactamase (pMM85) were grown in TMH overnight at 21°C, normalized, and then subcultured in TMH (without CaCl 2 ) and incubated at 21°C (gray columns) or 37°C (black columns) for an additional 4 h. Bacterial lysates (A) and culture supernatants (B) were incubated with fluorocillin for 4 h, and the fluorescence of each sample was measured. The means and standard errors for three experiments are indicated. Y. pestis KIM5 lacking β-lactamase fusion proteins and sterile TMH medium were used as background controls.

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Incubation, Fluorescence

    T3SS-dependent secretion of the Y. pestis effector protein YopM but not the Tc proteins YitA and YipA. Y. pestis KIM10(pUC19:: yitR ) and Y. pestis (pMM83) (expresses YopM–β-lactamase) with or without pCD1::kan (T3SS + or T3SS − ) were grown in TMH overnight at 21°C, normalized, and then subcultured into TMH with or without CaCl 2 and incubated at either 21°C or 37°C for an additional 4 h. Concentrated culture supernatants were analyzed by Western blotting using anti-YitA, anti-YipA, and anti-β-lactamase antibodies. Anti-GroEL was used to evaluate cellular lysis. Results are representative of three separate experiments.
    Figure Legend Snippet: T3SS-dependent secretion of the Y. pestis effector protein YopM but not the Tc proteins YitA and YipA. Y. pestis KIM10(pUC19:: yitR ) and Y. pestis (pMM83) (expresses YopM–β-lactamase) with or without pCD1::kan (T3SS + or T3SS − ) were grown in TMH overnight at 21°C, normalized, and then subcultured into TMH with or without CaCl 2 and incubated at either 21°C or 37°C for an additional 4 h. Concentrated culture supernatants were analyzed by Western blotting using anti-YitA, anti-YipA, and anti-β-lactamase antibodies. Anti-GroEL was used to evaluate cellular lysis. Results are representative of three separate experiments.

    Techniques Used: Incubation, Western Blot, Lysis

    9) Product Images from "The Role of a Second-Shell Residue in Modifying Substrate and Inhibitor Interactions in the SHV β-Lactamase: A Study of Ambler Position Asn276"

    Article Title: The Role of a Second-Shell Residue in Modifying Substrate and Inhibitor Interactions in the SHV β-Lactamase: A Study of Ambler Position Asn276

    Journal: Biochemistry

    doi: 10.1021/bi9003292

    Kinetic Behavior of Asn276Asp with β-Lactamase Inhibitors: Probes of the Active Site
    Figure Legend Snippet: Kinetic Behavior of Asn276Asp with β-Lactamase Inhibitors: Probes of the Active Site

    Techniques Used:

    10) Product Images from "Moraxella catarrhalis Outer Membrane Vesicles Carry ?-Lactamase and Promote Survival of Streptococcus pneumoniae and Haemophilus influenzae by Inactivating Amoxicillin ▿"

    Article Title: Moraxella catarrhalis Outer Membrane Vesicles Carry ?-Lactamase and Promote Survival of Streptococcus pneumoniae and Haemophilus influenzae by Inactivating Amoxicillin ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01772-10

    M. catarrhalis β-lactamase-containing OMVs protect S. pneumoniae (pneumococci [Pnc]) from being killed by amoxicillin. Amoxicillin-susceptible S. pneumoniae ATCC 6303 (10 6 CFU/ml) was grown with amoxicillin (1 μg/ml) preincubated with either 25 μg/ml β-lactamase-positive OMVs (A and B) or β-lactamase-negative OMVs (B and C). OMVs were isolated from M. catarrhalis KR526 (β-lac + ) and Bc5 (β-lac − ). Growth was expressed either as relative growth compared to starting concentrations measured as absorbance (OD 600 ) (A and C) or as numbers of CFU (B). The data are presented as means and the standard errors of at least three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
    Figure Legend Snippet: M. catarrhalis β-lactamase-containing OMVs protect S. pneumoniae (pneumococci [Pnc]) from being killed by amoxicillin. Amoxicillin-susceptible S. pneumoniae ATCC 6303 (10 6 CFU/ml) was grown with amoxicillin (1 μg/ml) preincubated with either 25 μg/ml β-lactamase-positive OMVs (A and B) or β-lactamase-negative OMVs (B and C). OMVs were isolated from M. catarrhalis KR526 (β-lac + ) and Bc5 (β-lac − ). Growth was expressed either as relative growth compared to starting concentrations measured as absorbance (OD 600 ) (A and C) or as numbers of CFU (B). The data are presented as means and the standard errors of at least three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Techniques Used: Isolation

    Vesicles from β-lactamase-positive M. catarrhalis protect NTHi against amoxicillin. Amoxicillin-susceptible NTHi 772 (A to C) or Hib KR124 (D) (10 7 CFU/ml) was grown with amoxicillin (2 μg/ml) that had been preincubated with either 25 μg/ml β-lactamase-positive OMVs (A and B) or β-lactamase-negative OMVs (B and C). OMVs were isolated from M. catarrhalis KR526 and Bc5, which were β-lactamase positive and negative, respectively. Growth was expressed either as relative growth compared to starting concentrations measured as absorbance at OD 600 (A and C) or as numbers of CFU (B and D). The results are shown as means and SEMs of at least three independent experiments. *, P ≤ 0.05; ***, P ≤ 0.001.
    Figure Legend Snippet: Vesicles from β-lactamase-positive M. catarrhalis protect NTHi against amoxicillin. Amoxicillin-susceptible NTHi 772 (A to C) or Hib KR124 (D) (10 7 CFU/ml) was grown with amoxicillin (2 μg/ml) that had been preincubated with either 25 μg/ml β-lactamase-positive OMVs (A and B) or β-lactamase-negative OMVs (B and C). OMVs were isolated from M. catarrhalis KR526 and Bc5, which were β-lactamase positive and negative, respectively. Growth was expressed either as relative growth compared to starting concentrations measured as absorbance at OD 600 (A and C) or as numbers of CFU (B and D). The results are shown as means and SEMs of at least three independent experiments. *, P ≤ 0.05; ***, P ≤ 0.001.

    Techniques Used: Isolation

    M. catarrhalis OMVs contain enzymatically active β-lactamase. (A) The β-lactamase contents of whole-cell lysates and OMVs from four different M. catarrhalis strains were analyzed. OMVs contained approximately the same β-lactamase content as whole-cell lysates. (B) The β-lactamase enzyme was found on the inside of the OMVs. The β-lactamase activity was quantified by the ability of the enzyme to hydrolyze the β-lactam nitrocefin, leading to a change in absorbance from OD 380 to OD 485 , as determined by spectrophotometry. β-Lactamase enzyme or OMVs were treated with proteinase K (100 μg/ml) and/or saponin (0.2%), and enzyme content was subsequently determined. As a negative control, OMVs were first treated with saponin, followed by proteinase K. In panel A, the β-lactamase content was expressed as the number of moles nitrocefin hydrolyzed per minute per mg OMVs. In panel B, β-lactamase activity was defined as the percentage of enzyme activity of OMV in the absence of amoxicillin that was set to 100%. Data shown are means and SEM of at least three independent experiments.
    Figure Legend Snippet: M. catarrhalis OMVs contain enzymatically active β-lactamase. (A) The β-lactamase contents of whole-cell lysates and OMVs from four different M. catarrhalis strains were analyzed. OMVs contained approximately the same β-lactamase content as whole-cell lysates. (B) The β-lactamase enzyme was found on the inside of the OMVs. The β-lactamase activity was quantified by the ability of the enzyme to hydrolyze the β-lactam nitrocefin, leading to a change in absorbance from OD 380 to OD 485 , as determined by spectrophotometry. β-Lactamase enzyme or OMVs were treated with proteinase K (100 μg/ml) and/or saponin (0.2%), and enzyme content was subsequently determined. As a negative control, OMVs were first treated with saponin, followed by proteinase K. In panel A, the β-lactamase content was expressed as the number of moles nitrocefin hydrolyzed per minute per mg OMVs. In panel B, β-lactamase activity was defined as the percentage of enzyme activity of OMV in the absence of amoxicillin that was set to 100%. Data shown are means and SEM of at least three independent experiments.

    Techniques Used: Activity Assay, Spectrophotometry, Negative Control

    Amoxicillin-resistant M. catarrhalis strains produce OMV containing β-lactamase. (A) Eight M. catarrhalis strains out of 10 were positive for the bro gene (522 bp), as revealed by PCR analysis. bro alleles were not found in strains KR395 and Bc5. (B) M. catarrhalis OMVs and total bacterial lysates (10 μg each) were subjected to SDS-PAGE (left), followed by detection of β-lactamase (35 kDa) by Western blotting (right). Lysates of whole M. catarrhalis RH4 and KR395 bacteria were used as positive and negative controls, respectively. Recombinant RH4 β-lactamase (0.6 μg) was also included. The upper band represents the recombinant protein at a size of 37.7 kDa. The lower band most likely results from N-terminal degradation. The His tag located at the C-terminal end was not affected by degradation, since it was possible to purify the recombinant β-lactamase using affinity chromatography. (C) Flow cytometry using antiserum raised against recombinant RH4 β-lactamase. The arrow shows a positive shift with β-lactamase containing OMV KR526, whereas OMVs from Bc5 were negative. OMVs without the β-lactamase antiserum (black) were compared to OMVs incubated with β-lactamase antiserum (white). (D) Gold-labeled anti-β-lactamase antibodies confirmed the presence of β-lactamase by TEM.
    Figure Legend Snippet: Amoxicillin-resistant M. catarrhalis strains produce OMV containing β-lactamase. (A) Eight M. catarrhalis strains out of 10 were positive for the bro gene (522 bp), as revealed by PCR analysis. bro alleles were not found in strains KR395 and Bc5. (B) M. catarrhalis OMVs and total bacterial lysates (10 μg each) were subjected to SDS-PAGE (left), followed by detection of β-lactamase (35 kDa) by Western blotting (right). Lysates of whole M. catarrhalis RH4 and KR395 bacteria were used as positive and negative controls, respectively. Recombinant RH4 β-lactamase (0.6 μg) was also included. The upper band represents the recombinant protein at a size of 37.7 kDa. The lower band most likely results from N-terminal degradation. The His tag located at the C-terminal end was not affected by degradation, since it was possible to purify the recombinant β-lactamase using affinity chromatography. (C) Flow cytometry using antiserum raised against recombinant RH4 β-lactamase. The arrow shows a positive shift with β-lactamase containing OMV KR526, whereas OMVs from Bc5 were negative. OMVs without the β-lactamase antiserum (black) were compared to OMVs incubated with β-lactamase antiserum (white). (D) Gold-labeled anti-β-lactamase antibodies confirmed the presence of β-lactamase by TEM.

    Techniques Used: Polymerase Chain Reaction, SDS Page, Western Blot, Recombinant, Affinity Chromatography, Flow Cytometry, Cytometry, Incubation, Labeling, Transmission Electron Microscopy

    β-Lactamase-carrying M. catarrhalis OMVs hydrolyze amoxicillin. (A) Amoxicillin (AMX)-induced killing at 1.25 to 10 μg/ml amoxicillin was gradually reduced with increasing concentrations (0 to 50 μg/ml) of β-lactamase-containing OMVs. Amoxicillin concentrations were determined by measuring inhibitory growth zones of the β-lactam-susceptible bacterium Sarcina lutea . (B) β-Lactamase-positive and -negative M. catarrhalis OMVs at 25 μg/ml were incubated with increasing amoxicillin concentrations. The data are presented as means and SEMs of at least three independent experiments. β-lac + and β-lac − , β-lactamase positive and negative, respectively. ***, P ≤ 0.001.
    Figure Legend Snippet: β-Lactamase-carrying M. catarrhalis OMVs hydrolyze amoxicillin. (A) Amoxicillin (AMX)-induced killing at 1.25 to 10 μg/ml amoxicillin was gradually reduced with increasing concentrations (0 to 50 μg/ml) of β-lactamase-containing OMVs. Amoxicillin concentrations were determined by measuring inhibitory growth zones of the β-lactam-susceptible bacterium Sarcina lutea . (B) β-Lactamase-positive and -negative M. catarrhalis OMVs at 25 μg/ml were incubated with increasing amoxicillin concentrations. The data are presented as means and SEMs of at least three independent experiments. β-lac + and β-lac − , β-lactamase positive and negative, respectively. ***, P ≤ 0.001.

    Techniques Used: Incubation

    β-Lactamase-positive M. catarrhalis OMVs protect amoxicillin-susceptible M. catarrhalis strains from being killed by amoxicillin. β-Lactamase-susceptible M. catarrhalis KR935 (10 7 CFU/ml) was grown with amoxicillin (1 μg/ml) that had been preincubated in the presence of 25 μg/ml β-lactamase-positive OMVs (A and B) or β-lactamase-negative OMVs (B and C). β-Lactamase-positive and -negative OMVs were isolated from M. catarrhalis KR526 (β-lac + ) and Bc5 (β-lac − ), respectively. Growth was expressed either as relative growth compared to starting concentrations measured as absorbance (OD 600 ) (A and C) or as numbers of CFU (B). Mean values and SEMs of at least three independent experiments are shown. *, P ≤ 0.05.
    Figure Legend Snippet: β-Lactamase-positive M. catarrhalis OMVs protect amoxicillin-susceptible M. catarrhalis strains from being killed by amoxicillin. β-Lactamase-susceptible M. catarrhalis KR935 (10 7 CFU/ml) was grown with amoxicillin (1 μg/ml) that had been preincubated in the presence of 25 μg/ml β-lactamase-positive OMVs (A and B) or β-lactamase-negative OMVs (B and C). β-Lactamase-positive and -negative OMVs were isolated from M. catarrhalis KR526 (β-lac + ) and Bc5 (β-lac − ), respectively. Growth was expressed either as relative growth compared to starting concentrations measured as absorbance (OD 600 ) (A and C) or as numbers of CFU (B). Mean values and SEMs of at least three independent experiments are shown. *, P ≤ 0.05.

    Techniques Used: Isolation

    11) Product Images from "Design and Exploration of Novel Boronic Acid Inhibitors Reveals Important Interactions with a Clavulanic Acid-Resistant Sulfhydryl-Variable (SHV) \u03b2-Lactamase"

    Article Title: Design and Exploration of Novel Boronic Acid Inhibitors Reveals Important Interactions with a Clavulanic Acid-Resistant Sulfhydryl-Variable (SHV) \u03b2-Lactamase

    Journal: Journal of medicinal chemistry

    doi: 10.1021/jm301490d

    The top figure shows the wild-type enzyme with Ser-130 at a χ angle of −144° where proton shuffling can occur to allow breakdown of ampicillin by the β-lactamase. The bottom figure shows the movement of the hydroxyl group
    Figure Legend Snippet: The top figure shows the wild-type enzyme with Ser-130 at a χ angle of −144° where proton shuffling can occur to allow breakdown of ampicillin by the β-lactamase. The bottom figure shows the movement of the hydroxyl group

    Techniques Used:

    Kinetics of K234R with β-Lactamase Inhibitors
    Figure Legend Snippet: Kinetics of K234R with β-Lactamase Inhibitors

    Techniques Used:

    12) Product Images from "Copper Influences the Antibacterial Outcomes of a β-Lactamase-Activated Prochelator against Drug-Resistant Bacteria"

    Article Title: Copper Influences the Antibacterial Outcomes of a β-Lactamase-Activated Prochelator against Drug-Resistant Bacteria

    Journal: ACS infectious diseases

    doi: 10.1021/acsinfecdis.8b00037

    Prochelator PcephPT inhibits growth of β-lactamase-expressing E. coli .
    Figure Legend Snippet: Prochelator PcephPT inhibits growth of β-lactamase-expressing E. coli .

    Techniques Used: Expressing

    Ability of PcephPT to kill MG1655 bacteria depends on both the presence of β-lactamase and availability of Cu.
    Figure Legend Snippet: Ability of PcephPT to kill MG1655 bacteria depends on both the presence of β-lactamase and availability of Cu.

    Techniques Used:

    Prochelator PcephPT is selectively turned over by bacteria that produce β-lactamase.
    Figure Legend Snippet: Prochelator PcephPT is selectively turned over by bacteria that produce β-lactamase.

    Techniques Used:

    13) Product Images from "Kinetic Properties of Four Plasmid-Mediated AmpC ?-Lactamases"

    Article Title: Kinetic Properties of Four Plasmid-Mediated AmpC ?-Lactamases

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.49.10.4240-4246.2005

    Titration curve of the ACT-1 β-lactamase by aztreonam. The residual activity of ACT-1 versus the [aztreonam]/[ACT-1] ratio is shown. The linear regression allows the determination of the actual concentrations of active enzyme. E th , theoretical
    Figure Legend Snippet: Titration curve of the ACT-1 β-lactamase by aztreonam. The residual activity of ACT-1 versus the [aztreonam]/[ACT-1] ratio is shown. The linear regression allows the determination of the actual concentrations of active enzyme. E th , theoretical

    Techniques Used: Titration, Activated Clotting Time Assay, Activity Assay

    14) Product Images from "Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway"

    Article Title: Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04985-0

    Human mo-DCs and mo-Mac are inefficient for the transfer of exogenous proteins into their cytosol. a , b Purified DCs and macrophages from tumor ascites, derived in vitro from monocytes, or DCs derived in vitro from CD34 + precursors were loaded with a cell-permeable FRET-sensitive substrate of β-lactamase, and incubated with or without exogenous β-lactamase. After 3 h, cleavage was measured by flow cytometry. a Representative results of six (tumor ascites), ten (in vitro monocyte-derived), or eight (in vitro CD34 + cell-derived) independent experiments. b Quantification of β-lactamase transfer. Symbols represent individual donors. N = 6 for tumor ascites, N = 10 for in vitro monocyte-derived cells, and N = 8 for CD34 + cell-derived cells. ** p
    Figure Legend Snippet: Human mo-DCs and mo-Mac are inefficient for the transfer of exogenous proteins into their cytosol. a , b Purified DCs and macrophages from tumor ascites, derived in vitro from monocytes, or DCs derived in vitro from CD34 + precursors were loaded with a cell-permeable FRET-sensitive substrate of β-lactamase, and incubated with or without exogenous β-lactamase. After 3 h, cleavage was measured by flow cytometry. a Representative results of six (tumor ascites), ten (in vitro monocyte-derived), or eight (in vitro CD34 + cell-derived) independent experiments. b Quantification of β-lactamase transfer. Symbols represent individual donors. N = 6 for tumor ascites, N = 10 for in vitro monocyte-derived cells, and N = 8 for CD34 + cell-derived cells. ** p

    Techniques Used: Purification, Derivative Assay, In Vitro, Incubation, Flow Cytometry, Cytometry

    15) Product Images from "Nosocomial Spread of Ceftazidime-Resistant Klebsiella pneumoniae Strains Producing a Novel Class A ?-Lactamase, GES-3, in a Neonatal Intensive Care Unit in Japan"

    Article Title: Nosocomial Spread of Ceftazidime-Resistant Klebsiella pneumoniae Strains Producing a Novel Class A ?-Lactamase, GES-3, in a Neonatal Intensive Care Unit in Japan

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.6.1960-1967.2004

    IEF. (A) IEF and staining with Coomassie blue. Lanes: M, pI marker; GES-3, purified GES-3 enzyme. (B) IEF and staining with nitrocefin. Lanes: 1, GES-3-producing E. coli transformant that harbors pKGB525 carrying bla GES-3 ; 2, K. pneumoniae KG914; 3, K. pneumoniae KG1116; 4, K. pneumoniae KG525; 5, K. pneumoniae KG545; 6, K. pneumoniae KG502; 7, K. pneumoniae KG827. The bands of pI 7.63 (pI 7.6 in the text) are the chromosomally encoded LEN-1 or SHV-1 β-lactamase of K. pneumoniae , and the bands at pI 6.98 (pI 7.0 in the text) are GES-3. Several β-lactamases with activities at pIs lower than 7.0 were speculated to be partially unfolded GES-3 β-lactamase, because these bands were also found in the IEF gels of an E. coli clone harboring only the bla GES-3 gene (data not shown).
    Figure Legend Snippet: IEF. (A) IEF and staining with Coomassie blue. Lanes: M, pI marker; GES-3, purified GES-3 enzyme. (B) IEF and staining with nitrocefin. Lanes: 1, GES-3-producing E. coli transformant that harbors pKGB525 carrying bla GES-3 ; 2, K. pneumoniae KG914; 3, K. pneumoniae KG1116; 4, K. pneumoniae KG525; 5, K. pneumoniae KG545; 6, K. pneumoniae KG502; 7, K. pneumoniae KG827. The bands of pI 7.63 (pI 7.6 in the text) are the chromosomally encoded LEN-1 or SHV-1 β-lactamase of K. pneumoniae , and the bands at pI 6.98 (pI 7.0 in the text) are GES-3. Several β-lactamases with activities at pIs lower than 7.0 were speculated to be partially unfolded GES-3 β-lactamase, because these bands were also found in the IEF gels of an E. coli clone harboring only the bla GES-3 gene (data not shown).

    Techniques Used: Electrofocusing, Staining, Marker, Purification

    16) Product Images from "AnkB, a Periplasmic Ankyrin-Like Protein in Pseudomonas aeruginosa, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide"

    Article Title: AnkB, a Periplasmic Ankyrin-Like Protein in Pseudomonas aeruginosa, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide

    Journal: Journal of Bacteriology

    doi:

    Cellular localization of AnkB in P. aeruginosa . (A) Predicted cytoplasmic membrane organization of P. aeruginosa AnkB bacterial ankyrin-like proteins from P. syringae , P. fluorescens , S. liquefaciens , and C. vinosum ). For the P. aeruginosa AnkB protein, the large number 1 indicates the predicted single MSD. N, N terminus; C, C terminus; LL, loop length; KR, number of lysine and arginine residues; KR Diff, positive charge difference. (B) Schematic diagram of AnkB–β-lactamase and AnkB-PhoA protein fusions in both E. coli and P. aeruginosa PAO1. In both cases, organisms expressing AnkB–β-lactamase were resistant to ampicillin ( E. coli ) or carbenicillin ( P. aeruginosa ). Organisms expressing AnkB-PhoA were found to hydrolyze the alkaline phosphatase substrate BCIP in L-agar plates. IM, inner membrane; OM, outer membrane. (C) AP activity in cellular fractions of P. aeruginosa ankB harboring pEX30- ankB :: phoA . Bar 1, cytoplasm; bar 2, periplasm; bar 3, cytoplasmic membrane; bar 4, outer membrane.
    Figure Legend Snippet: Cellular localization of AnkB in P. aeruginosa . (A) Predicted cytoplasmic membrane organization of P. aeruginosa AnkB bacterial ankyrin-like proteins from P. syringae , P. fluorescens , S. liquefaciens , and C. vinosum ). For the P. aeruginosa AnkB protein, the large number 1 indicates the predicted single MSD. N, N terminus; C, C terminus; LL, loop length; KR, number of lysine and arginine residues; KR Diff, positive charge difference. (B) Schematic diagram of AnkB–β-lactamase and AnkB-PhoA protein fusions in both E. coli and P. aeruginosa PAO1. In both cases, organisms expressing AnkB–β-lactamase were resistant to ampicillin ( E. coli ) or carbenicillin ( P. aeruginosa ). Organisms expressing AnkB-PhoA were found to hydrolyze the alkaline phosphatase substrate BCIP in L-agar plates. IM, inner membrane; OM, outer membrane. (C) AP activity in cellular fractions of P. aeruginosa ankB harboring pEX30- ankB :: phoA . Bar 1, cytoplasm; bar 2, periplasm; bar 3, cytoplasmic membrane; bar 4, outer membrane.

    Techniques Used: Expressing, Activity Assay

    17) Product Images from "Design and Exploration of Novel Boronic Acid Inhibitors Reveals Important Interactions with a Clavulanic Acid-Resistant Sulfhydryl-Variable (SHV) \u03b2-Lactamase"

    Article Title: Design and Exploration of Novel Boronic Acid Inhibitors Reveals Important Interactions with a Clavulanic Acid-Resistant Sulfhydryl-Variable (SHV) \u03b2-Lactamase

    Journal: Journal of medicinal chemistry

    doi: 10.1021/jm301490d

    The top figure shows the wild-type enzyme with Ser-130 at a χ angle of −144° where proton shuffling can occur to allow breakdown of ampicillin by the β-lactamase. The bottom figure shows the movement of the hydroxyl group
    Figure Legend Snippet: The top figure shows the wild-type enzyme with Ser-130 at a χ angle of −144° where proton shuffling can occur to allow breakdown of ampicillin by the β-lactamase. The bottom figure shows the movement of the hydroxyl group

    Techniques Used:

    Kinetics of K234R with β-Lactamase Inhibitors
    Figure Legend Snippet: Kinetics of K234R with β-Lactamase Inhibitors

    Techniques Used:

    18) Product Images from "Molecular Characterization of Chromosomal Class C ?-Lactamase and Its Regulatory Gene in Ochrobactrum anthropi"

    Article Title: Molecular Characterization of Chromosomal Class C ?-Lactamase and Its Regulatory Gene in Ochrobactrum anthropi

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.45.8.2324-2330.2001

    Alignment of the ampC-ampR intercistronic region from the β-lactamase of the following strains: 1, O. anthropi SLO74; 2, P. aeruginosa PAO01; 3, P. stuartii VDG 96; 4, M. morganii SLM 01; 5, Y. enterocolitica IP 97; 6, C. freundii OS 60. The Lys-R motif is boxed.
    Figure Legend Snippet: Alignment of the ampC-ampR intercistronic region from the β-lactamase of the following strains: 1, O. anthropi SLO74; 2, P. aeruginosa PAO01; 3, P. stuartii VDG 96; 4, M. morganii SLM 01; 5, Y. enterocolitica IP 97; 6, C. freundii OS 60. The Lys-R motif is boxed.

    Techniques Used:

    19) Product Images from "Application of termite hindgut metagenome derived carboxyl ester hydrolases in the modification of cephalosporin substrates"

    Article Title: Application of termite hindgut metagenome derived carboxyl ester hydrolases in the modification of cephalosporin substrates

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2015.08.016

    HPLC analysis of the cephalosporin derivatives reactions performed with AxeA and AxeB using β-lactamase as the positive control, (A) Cephalothin substrate and (B) 7-amino-cephalosporanic acid. Substrates only were also analysed with the reaction products.
    Figure Legend Snippet: HPLC analysis of the cephalosporin derivatives reactions performed with AxeA and AxeB using β-lactamase as the positive control, (A) Cephalothin substrate and (B) 7-amino-cephalosporanic acid. Substrates only were also analysed with the reaction products.

    Techniques Used: High Performance Liquid Chromatography, Positive Control

    20) Product Images from "Controlling resistant bacteria with a novel class of β-lactamase inhibitor peptides: from rational design to in vivo analyses"

    Article Title: Controlling resistant bacteria with a novel class of β-lactamase inhibitor peptides: from rational design to in vivo analyses

    Journal: Scientific Reports

    doi: 10.1038/srep06015

    Binding and inhibitory activity of Bacillus cereus 569 β-lactamase (Calbiochem, UK) by using dBLIP-1 and -2. MALDI-ToF Mass spectra of purified dBLIP-1 (a) and dBLIP-2 (b). In vitro study for inhibition (IC50) of β-lactamase activity by dBLIP-1 and dBLIP-2, (c). ITC bonds measurement between dBLIP-1 and 2. Figures show the raw data obtained from ITC binding experiment between β-lactamase with dBLIP-1 (d) and dBLIP-2 (f). Figure representing the integration of raw heat associated data for dBLIP-1 (e) and dBLIP-2 (g). These data have been corrected by subtraction of appropriate blank experiments and then fitted with nonlinear regression; data derived after analysis have been listed in Supplementary Table 1 . The binding thermodynamics for these experiments are listed in the methods section.
    Figure Legend Snippet: Binding and inhibitory activity of Bacillus cereus 569 β-lactamase (Calbiochem, UK) by using dBLIP-1 and -2. MALDI-ToF Mass spectra of purified dBLIP-1 (a) and dBLIP-2 (b). In vitro study for inhibition (IC50) of β-lactamase activity by dBLIP-1 and dBLIP-2, (c). ITC bonds measurement between dBLIP-1 and 2. Figures show the raw data obtained from ITC binding experiment between β-lactamase with dBLIP-1 (d) and dBLIP-2 (f). Figure representing the integration of raw heat associated data for dBLIP-1 (e) and dBLIP-2 (g). These data have been corrected by subtraction of appropriate blank experiments and then fitted with nonlinear regression; data derived after analysis have been listed in Supplementary Table 1 . The binding thermodynamics for these experiments are listed in the methods section.

    Techniques Used: Binding Assay, Activity Assay, Purification, In Vitro, Inhibition, Derivative Assay

    Docking studies of dBLIP-1 and dBLIP-2 and β-lactamases from two different bacterial sources. Structural complementarity between dBLIP-1 (a) and dBLIP-2 (b) toward β-lactamase from Escherichia coli . In detail, the non-covalent interactions (dotted lines) of dBLIP-1 (c) and dBLIP-2 (D) and E. coli β-lactamase catalytic site. Structural complementarity between dBLIP-1 (a) and dBLIP-2 (b) with β-lactamase from Staphylococcus aureus . In detail, the non-covalent interactions (dotted lines) of dBLIP-1 (c) and dBLIP-2 (d) and S. aureus β-lactamase catalytic site.
    Figure Legend Snippet: Docking studies of dBLIP-1 and dBLIP-2 and β-lactamases from two different bacterial sources. Structural complementarity between dBLIP-1 (a) and dBLIP-2 (b) toward β-lactamase from Escherichia coli . In detail, the non-covalent interactions (dotted lines) of dBLIP-1 (c) and dBLIP-2 (D) and E. coli β-lactamase catalytic site. Structural complementarity between dBLIP-1 (a) and dBLIP-2 (b) with β-lactamase from Staphylococcus aureus . In detail, the non-covalent interactions (dotted lines) of dBLIP-1 (c) and dBLIP-2 (d) and S. aureus β-lactamase catalytic site.

    Techniques Used:

    21) Product Images from "Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export"

    Article Title: Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1705242114

    Acute inhibition of Sec61 with mycolactone (ML) does not inhibit antigen export or cross-presentation. ( A ) ML efficiently prevents the TLR4- or TLR3-induced production of Sec61 substrates in MutuDCs. Cells were activated by 0.5 μg/mL LPS or 5 μg/mL high-molecular-weight poly(I:C) and incubated in the presence of 100 nM ML for 16 h. CTR, control; MFI, mean fluorescence intensity. Up-regulation of CD86 surface expression was monitored by flow cytometry. ( B ) Schematic representation of changes in CCF4 fluorescence upon cleavage of the the β-lactam (β-lac) ring. ( C ) Schematic representation of the antigen export assay. MutuDCs are fed with the β-lactamase and loaded with CCF4 in B , and the efficiency of CCF4 cleavage is analyzed by flow cytometry. ( D ) MutuDCs were incubated with β-lac in the presence or absence of increasing concentrations of ML for 3 h and analyzed as described in C . ( E ) Schematic representation of the cross-presentation assay. ( F ) MutuDCs were incubated for 5 h with soluble OVA protein or OVA 257–264 peptide in the presence or absence of increasing concentrations of ML. DCs were then fixed and coincubated with B3Z hybridomas for 16 h. Up-regulation of β-galactosidase (β-gal) expression in B3Zs (driven by the IL-2 promoter) was quantified using a colorimetric substrate, CPRG. For D and F , representative data from three independent experiments are shown.
    Figure Legend Snippet: Acute inhibition of Sec61 with mycolactone (ML) does not inhibit antigen export or cross-presentation. ( A ) ML efficiently prevents the TLR4- or TLR3-induced production of Sec61 substrates in MutuDCs. Cells were activated by 0.5 μg/mL LPS or 5 μg/mL high-molecular-weight poly(I:C) and incubated in the presence of 100 nM ML for 16 h. CTR, control; MFI, mean fluorescence intensity. Up-regulation of CD86 surface expression was monitored by flow cytometry. ( B ) Schematic representation of changes in CCF4 fluorescence upon cleavage of the the β-lactam (β-lac) ring. ( C ) Schematic representation of the antigen export assay. MutuDCs are fed with the β-lactamase and loaded with CCF4 in B , and the efficiency of CCF4 cleavage is analyzed by flow cytometry. ( D ) MutuDCs were incubated with β-lac in the presence or absence of increasing concentrations of ML for 3 h and analyzed as described in C . ( E ) Schematic representation of the cross-presentation assay. ( F ) MutuDCs were incubated for 5 h with soluble OVA protein or OVA 257–264 peptide in the presence or absence of increasing concentrations of ML. DCs were then fixed and coincubated with B3Z hybridomas for 16 h. Up-regulation of β-galactosidase (β-gal) expression in B3Zs (driven by the IL-2 promoter) was quantified using a colorimetric substrate, CPRG. For D and F , representative data from three independent experiments are shown.

    Techniques Used: Inhibition, Molecular Weight, Incubation, Fluorescence, Expressing, Flow Cytometry, Cytometry

    22) Product Images from "VAMP8-mediated NOX2 recruitment to endosomes is necessary for antigen release"

    Article Title: VAMP8-mediated NOX2 recruitment to endosomes is necessary for antigen release

    Journal: European Journal of Cell Biology

    doi: 10.1016/j.ejcb.2017.06.007

    Antigen cross-presentation depends on VAMP8-mediated trafficking of NOX2. (A) Representative confocal microscope images of the CCF4 endosomal leakage assay for VAMP8 siRNA knockdown (VAMP8 KD) and non-targeting siRNA (NT) human DCs. The cytosolic FRET probe CCF4 was cleaved by exogenous β-lactamase (β-lac) resulting in a decreased ratio of fluorescein (acceptor fluorophore; green in merge) over coumarin (donor; blue) fluorescence. (B) Representative confocal microscope images as in panel A, but now for VAMP8-/- and VAMP8± mouse BMDCs. (C) Quantification of panel A. The graph shows the CCF4 cleavage efficiencies (see reference ( Dingjan et al., 2016 )) of DCs from 3 donors (linked by solid lines; p = 0.0312). (D) Same as panel C, but now for VAMP8-/- and VAMP8± mouse BMDCs (panel B; p = 0.0457). (E) Representative flow cytometry histograms of CD69 expression by Jurkat cells carrying a gp100-specific T cell receptor. The jurkat T cells were co-cultured with VAMP8 KD (blue) and NT (green) DCs that were loaded with short (residues 280–288; left-hand graphs) or long (residues 272–300; right) gp100 peptide. The percentages CD69-positive T cells are indicated in the graphs. (F) Quantification of T cell activation from panel E for 4 donors (p = 0.0461). Scale bars, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Antigen cross-presentation depends on VAMP8-mediated trafficking of NOX2. (A) Representative confocal microscope images of the CCF4 endosomal leakage assay for VAMP8 siRNA knockdown (VAMP8 KD) and non-targeting siRNA (NT) human DCs. The cytosolic FRET probe CCF4 was cleaved by exogenous β-lactamase (β-lac) resulting in a decreased ratio of fluorescein (acceptor fluorophore; green in merge) over coumarin (donor; blue) fluorescence. (B) Representative confocal microscope images as in panel A, but now for VAMP8-/- and VAMP8± mouse BMDCs. (C) Quantification of panel A. The graph shows the CCF4 cleavage efficiencies (see reference ( Dingjan et al., 2016 )) of DCs from 3 donors (linked by solid lines; p = 0.0312). (D) Same as panel C, but now for VAMP8-/- and VAMP8± mouse BMDCs (panel B; p = 0.0457). (E) Representative flow cytometry histograms of CD69 expression by Jurkat cells carrying a gp100-specific T cell receptor. The jurkat T cells were co-cultured with VAMP8 KD (blue) and NT (green) DCs that were loaded with short (residues 280–288; left-hand graphs) or long (residues 272–300; right) gp100 peptide. The percentages CD69-positive T cells are indicated in the graphs. (F) Quantification of T cell activation from panel E for 4 donors (p = 0.0461). Scale bars, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Microscopy, Fluorescence, Flow Cytometry, Cytometry, Expressing, Cell Culture, Activation Assay

    23) Product Images from "Immune Responses to Recombinant Pneumococcal PspA Antigen Delivered by Live Attenuated Salmonella enterica Serovar Typhimurium Vaccine "

    Article Title: Immune Responses to Recombinant Pneumococcal PspA Antigen Delivered by Live Attenuated Salmonella enterica Serovar Typhimurium Vaccine

    Journal: Infection and Immunity

    doi: 10.1128/IAI.70.4.1739-1749.2002

    Asd + antigen expression vectors. (A) Asd + vector pYA3342. The map of pYA3342 and the nucleotide sequences of the P trc promoter region and multicloning sites are shown. (B) Periplasmic secretion Asd + vector pYA3493. A DNA fragment encoding the β-lactamase signal sequence and 12 amino acid residues of the N terminus of mature β-lactamase of plasmid pBR322 was positioned under the control of the P trc promoter of the Asd + vector pYA3342 (pBR ori ). The map of pYA3493 and the nucleotide sequences of the P trc promoter region, β-lactamase signal sequence ( bla SS), and multicloning sites are shown. The P trc sequences for −35, −10, and SD are indicated, and the translation start codon is in boldface. An arrow within the sequence indicates the signal peptidase cleavage site. Unique restriction enzyme sites in the multicloning site are indicated. 5ST1T2 is a transcriptional terminator.
    Figure Legend Snippet: Asd + antigen expression vectors. (A) Asd + vector pYA3342. The map of pYA3342 and the nucleotide sequences of the P trc promoter region and multicloning sites are shown. (B) Periplasmic secretion Asd + vector pYA3493. A DNA fragment encoding the β-lactamase signal sequence and 12 amino acid residues of the N terminus of mature β-lactamase of plasmid pBR322 was positioned under the control of the P trc promoter of the Asd + vector pYA3342 (pBR ori ). The map of pYA3493 and the nucleotide sequences of the P trc promoter region, β-lactamase signal sequence ( bla SS), and multicloning sites are shown. The P trc sequences for −35, −10, and SD are indicated, and the translation start codon is in boldface. An arrow within the sequence indicates the signal peptidase cleavage site. Unique restriction enzyme sites in the multicloning site are indicated. 5ST1T2 is a transcriptional terminator.

    Techniques Used: Expressing, Plasmid Preparation, Sequencing

    24) Product Images from "Phage-displayed combinatorial peptide libraries in fusion to ?-lactamase as reporter for an accelerated clone screening: Potential uses of selected enzyme-linked affinity reagents in downstream applications"

    Article Title: Phage-displayed combinatorial peptide libraries in fusion to ?-lactamase as reporter for an accelerated clone screening: Potential uses of selected enzyme-linked affinity reagents in downstream applications

    Journal: Combinatorial chemistry & high throughput screening

    doi:

    Fluorescence imaging of ErbB2 over-expressing cells for their binding to ErbB2 ECD positive clones The figure shows binding of representative clones 113 and 150 to MCF-7/ErbB2 and SK-BR-3 cells, which are known to over-express the ErbB2 receptor. High intensities of fluorescence are seen in both MCF-7/ErbB2 and SK-BR-3 cells in comparison to minimally expressive MCF-7 cells. Negative controls of wild-type β-lactamase (Wt-BLA) and unselected library clones did not show higher fluorescence in MCF-7/ErbB2 and SK-BR-3 cells. The precipitating green fluorescence is produced by the β-lactamase activity associated with clones.
    Figure Legend Snippet: Fluorescence imaging of ErbB2 over-expressing cells for their binding to ErbB2 ECD positive clones The figure shows binding of representative clones 113 and 150 to MCF-7/ErbB2 and SK-BR-3 cells, which are known to over-express the ErbB2 receptor. High intensities of fluorescence are seen in both MCF-7/ErbB2 and SK-BR-3 cells in comparison to minimally expressive MCF-7 cells. Negative controls of wild-type β-lactamase (Wt-BLA) and unselected library clones did not show higher fluorescence in MCF-7/ErbB2 and SK-BR-3 cells. The precipitating green fluorescence is produced by the β-lactamase activity associated with clones.

    Techniques Used: Fluorescence, Imaging, Expressing, Binding Assay, Clone Assay, Produced, Activity Assay

    Binding of selected ErbB2 ECD positive clones to ErbB2 over-expressing cells The clones that were found to bind purified ErbB2 ECD were studied for their binding to ErbB2 over-expressing MCF-7/ErbB2 and SK-BR-3 cell-lines. The MCF-7 cell-line that is known to exhibit minimal ErbB2 expression was used for negative control cells. The screening was based on assaying the cell-bound β-lactamase activities. The clones expressing wild-type β-lactamase (Wt-BLA; without fusion peptide) and unselected library were used as negative clone controls. The bars represent β-lactamase activity as change in relative fluorescence unit (RFU)/min ± SE. *p≤0.05, significance of difference in the binding of clones to MCF-7 in comparison to MCF-7/ErbB2 or SK-BR-3 cells were determined using Student’s T-test. ErbB2 ECD , extracellular domain of ErbB2.
    Figure Legend Snippet: Binding of selected ErbB2 ECD positive clones to ErbB2 over-expressing cells The clones that were found to bind purified ErbB2 ECD were studied for their binding to ErbB2 over-expressing MCF-7/ErbB2 and SK-BR-3 cell-lines. The MCF-7 cell-line that is known to exhibit minimal ErbB2 expression was used for negative control cells. The screening was based on assaying the cell-bound β-lactamase activities. The clones expressing wild-type β-lactamase (Wt-BLA; without fusion peptide) and unselected library were used as negative clone controls. The bars represent β-lactamase activity as change in relative fluorescence unit (RFU)/min ± SE. *p≤0.05, significance of difference in the binding of clones to MCF-7 in comparison to MCF-7/ErbB2 or SK-BR-3 cells were determined using Student’s T-test. ErbB2 ECD , extracellular domain of ErbB2.

    Techniques Used: Binding Assay, Clone Assay, Expressing, Purification, Negative Control, Activity Assay, Fluorescence

    25) Product Images from "Display of Functional ?-Lactamase Inhibitory Protein on the Surface of M13 Bacteriophage"

    Article Title: Display of Functional ?-Lactamase Inhibitory Protein on the Surface of M13 Bacteriophage

    Journal: Antimicrobial Agents and Chemotherapy

    doi:

    Two-step phage ELISA results. (A) Binding curve to determine the subsaturation concentration of phage to use in step 2. A total of 1.3 × 10 11 phage were used in step 2. (B) Plot showing ELISA measurements of pG3-BLIP phage binding to immobilized β-lactamase. The IC 50 given shows the concentration of competing β-lactamase that results in half-maximal binding to the phagemid.
    Figure Legend Snippet: Two-step phage ELISA results. (A) Binding curve to determine the subsaturation concentration of phage to use in step 2. A total of 1.3 × 10 11 phage were used in step 2. (B) Plot showing ELISA measurements of pG3-BLIP phage binding to immobilized β-lactamase. The IC 50 given shows the concentration of competing β-lactamase that results in half-maximal binding to the phagemid.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

    26) Product Images from "Combinatorial microfluidic droplet engineering for biomimetic material synthesis"

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis

    Journal: Science Advances

    doi: 10.1126/sciadv.1600567

    Mineral shells formed from [W/O/W] double emulsions support in vitro protein expression. ( A ) Schematic overview of the second, double emulsion–producing, flow-focusing junction of a two-junction flow-focusing microfluidic device used to generate mineralized double emulsions (see fig. S10 and movie S4). ( B ) Schematic of [W/O/W] droplet mineralization to form an interfacial mineral layer around bacterial extracts in the innermost water phase. Here, “S” is a fluorogenic substrate (CCF2) that changes emission properties when enzymatically converted to product (“P”) by β-lactamase (β-lac) enzyme. ( C ) Polarized light optical micrograph shows interfacial birefringence. ( D ) Flow cytometry scatter plots of 50,000 droplets, proving that mineralized droplets support compartmentalized in vitro protein expression.
    Figure Legend Snippet: Mineral shells formed from [W/O/W] double emulsions support in vitro protein expression. ( A ) Schematic overview of the second, double emulsion–producing, flow-focusing junction of a two-junction flow-focusing microfluidic device used to generate mineralized double emulsions (see fig. S10 and movie S4). ( B ) Schematic of [W/O/W] droplet mineralization to form an interfacial mineral layer around bacterial extracts in the innermost water phase. Here, “S” is a fluorogenic substrate (CCF2) that changes emission properties when enzymatically converted to product (“P”) by β-lactamase (β-lac) enzyme. ( C ) Polarized light optical micrograph shows interfacial birefringence. ( D ) Flow cytometry scatter plots of 50,000 droplets, proving that mineralized droplets support compartmentalized in vitro protein expression.

    Techniques Used: In Vitro, Expressing, Flow Cytometry, Cytometry

    27) Product Images from "Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway"

    Article Title: Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04985-0

    Human mo-DCs and mo-Mac are inefficient for the transfer of exogenous proteins into their cytosol. a , b Purified DCs and macrophages from tumor ascites, derived in vitro from monocytes, or DCs derived in vitro from CD34 + precursors were loaded with a cell-permeable FRET-sensitive substrate of β-lactamase, and incubated with or without exogenous β-lactamase. After 3 h, cleavage was measured by flow cytometry. a Representative results of six (tumor ascites), ten (in vitro monocyte-derived), or eight (in vitro CD34 + cell-derived) independent experiments. b Quantification of β-lactamase transfer. Symbols represent individual donors. N = 6 for tumor ascites, N = 10 for in vitro monocyte-derived cells, and N = 8 for CD34 + cell-derived cells. ** p
    Figure Legend Snippet: Human mo-DCs and mo-Mac are inefficient for the transfer of exogenous proteins into their cytosol. a , b Purified DCs and macrophages from tumor ascites, derived in vitro from monocytes, or DCs derived in vitro from CD34 + precursors were loaded with a cell-permeable FRET-sensitive substrate of β-lactamase, and incubated with or without exogenous β-lactamase. After 3 h, cleavage was measured by flow cytometry. a Representative results of six (tumor ascites), ten (in vitro monocyte-derived), or eight (in vitro CD34 + cell-derived) independent experiments. b Quantification of β-lactamase transfer. Symbols represent individual donors. N = 6 for tumor ascites, N = 10 for in vitro monocyte-derived cells, and N = 8 for CD34 + cell-derived cells. ** p

    Techniques Used: Purification, Derivative Assay, In Vitro, Incubation, Flow Cytometry, Cytometry

    Related Articles

    Clone Assay:

    Article Title: Inhibition of the Class C ?-Lactamase from Acinetobacter spp: Insights into Effective Inhibitor Design †
    Article Snippet: .. For large-scale protein expression and β-lactamase characterization, the bla ADC gene (specifically, bla ADC-7 ) was cloned into pET24a (+) vector (kanamycin resistant, Novagen, Madison, WI) following a previously published method ( ). .. After sequencing verification, the correct construct was maintained in E. coli DH10B cells and transformed into E. coli BL21(DE3) cells for protein expression.

    Flow Cytometry:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Amplification:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Fluorescence:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Beta Lactamase Activity Assay:

    Article Title: Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and β-Lactamase Activity in Bacillus anthracis
    Article Snippet: .. Broth cultures in the late-exponential-growth phase (105 CFU/ml) of B. anthracis strains SK57, Ba3027, Ba0878, Sterne (PEN-S, select agent-excluded strain), and UT308 (PEN-R, select agent-excluded derivative of strain 32) were tested for β-lactamase activity using a nitrocefin-based quantitative β-lactamase activity assay (β-lactamase activity assay kit, MAK221; Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. .. Supernatants from 500 µl of culture from each strain were collected by centrifugation at 8,000 × g for 2 min through a 0.1-μm-pore-size polyvinylidene difluoride (PVDF) Ultrafree-MC spin-filter column (Millipore, Billerica, MA, USA).

    Cytometry:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Purification:

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli
    Article Snippet: .. The quality of the purified periplasmic fractions was tested by Western blot assay using anti-GroEL (kindly donated by Mario Cancino) and anti-β-lactamase (Chemicon, Temecula, CA) antibodies to detect GroEL (a cytoplasmic protein) and β-lactamase (a periplasmic protein), respectively. ..

    Produced:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Concentration Assay:

    Article Title: Binding Properties of a Peptide Derived from ?-Lactamase Inhibitory Protein
    Article Snippet: .. The β-lactamase gene was induced by adding isopropyl-β- d -thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and further incubation at 25°C for 4 h. Following induction the cells were pelleted and the supernatant containing the secreted, soluble β-lactamase was concentrated to 100 ml with an Amicon Centriprep-10 concentrator (Millipore Corp.). ..

    Incubation:

    Article Title: Binding Properties of a Peptide Derived from ?-Lactamase Inhibitory Protein
    Article Snippet: .. The β-lactamase gene was induced by adding isopropyl-β- d -thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and further incubation at 25°C for 4 h. Following induction the cells were pelleted and the supernatant containing the secreted, soluble β-lactamase was concentrated to 100 ml with an Amicon Centriprep-10 concentrator (Millipore Corp.). ..

    Activity Assay:

    Article Title: Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients
    Article Snippet: .. To assay β-lactamase activity in culture supernatants, P. aeruginosa cultures grown overnight with LB broth were passed through a filter (0.22 µm pore size, Millipore), and the cell-free supernatants were assayed for β-lactamase activity 30 min after addition of nitrocefin (Calbiochem), as described by the manufacturer, except that A490 was measured, and corrected by subtracting the value measured for the supernatant without nitrocefin. .. To determine the proportion of β-lactamase activity that is cell-associated, P. aeruginosa cultures grown overnight with LB broth were diluted 1:100 in LB broth and grown for 12 h. These cultures were centrifuged and the supernatant saved.

    Article Title: Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and β-Lactamase Activity in Bacillus anthracis
    Article Snippet: .. Broth cultures in the late-exponential-growth phase (105 CFU/ml) of B. anthracis strains SK57, Ba3027, Ba0878, Sterne (PEN-S, select agent-excluded strain), and UT308 (PEN-R, select agent-excluded derivative of strain 32) were tested for β-lactamase activity using a nitrocefin-based quantitative β-lactamase activity assay (β-lactamase activity assay kit, MAK221; Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. .. Supernatants from 500 µl of culture from each strain were collected by centrifugation at 8,000 × g for 2 min through a 0.1-μm-pore-size polyvinylidene difluoride (PVDF) Ultrafree-MC spin-filter column (Millipore, Billerica, MA, USA).

    Article Title: Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria
    Article Snippet: .. After 60 min, GEM particles and insoluble amylose azure were spun down, and the absorbance at 595 nm was determined. β-Lactamase activity was measured by adding 40 μl nitrocefin (CalBiochem) to GEM particles loaded with β-PA in 1 ml (final volume) of PBS. .. For immunofluorescence microscopy, suspensions containing 100 μl of GEM particles incubated with a PA fusion were washed twice with demineralized water and resuspended in an equal volume of PBS containing 1% BSA and mouse anti-c-myc antibody that was diluted 1:50.

    Expressing:

    Article Title: Inhibition of the Class C ?-Lactamase from Acinetobacter spp: Insights into Effective Inhibitor Design †
    Article Snippet: .. For large-scale protein expression and β-lactamase characterization, the bla ADC gene (specifically, bla ADC-7 ) was cloned into pET24a (+) vector (kanamycin resistant, Novagen, Madison, WI) following a previously published method ( ). .. After sequencing verification, the correct construct was maintained in E. coli DH10B cells and transformed into E. coli BL21(DE3) cells for protein expression.

    Western Blot:

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli
    Article Snippet: .. The quality of the purified periplasmic fractions was tested by Western blot assay using anti-GroEL (kindly donated by Mario Cancino) and anti-β-lactamase (Chemicon, Temecula, CA) antibodies to detect GroEL (a cytoplasmic protein) and β-lactamase (a periplasmic protein), respectively. ..

    Plasmid Preparation:

    Article Title: Inhibition of the Class C ?-Lactamase from Acinetobacter spp: Insights into Effective Inhibitor Design †
    Article Snippet: .. For large-scale protein expression and β-lactamase characterization, the bla ADC gene (specifically, bla ADC-7 ) was cloned into pET24a (+) vector (kanamycin resistant, Novagen, Madison, WI) following a previously published method ( ). .. After sequencing verification, the correct construct was maintained in E. coli DH10B cells and transformed into E. coli BL21(DE3) cells for protein expression.

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    Millipore β lactamase activity
    Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and <t>β-lactamase</t>
    β Lactamase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β lactamase activity/product/Millipore
    Average 95 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    β lactamase activity - by Bioz Stars, 2020-07
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    91
    Millipore lactamase activity
    Reporter assay for detection of AmpG permease activity. The <t>β-lactamase</t> assay was performed on Escherichia coli strains BW25113 (parental strain), TP73 (Δ ampD )/pNU305, AR74 (Δ ampG/ Δ ampD )/pNU305/ pACY-AR1 (empty vector), and AR74/pNU305/pAC-Tanf_08635. Activity was determined by chromogenic nitrocefin substrate induced at 0, 30, 60 min and absorbance was measured at OD 486 . Data are representative of three independent experiments with similar results. Each value represents the mean (±SD) of three values measured in one representative assay.
    Lactamase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lactamase activity/product/Millipore
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    lactamase activity - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    Image Search Results


    Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and β-lactamase

    Journal: Applied and Environmental Microbiology

    Article Title: Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

    doi: 10.1128/AEM.72.1.880-889.2006

    Figure Lengend Snippet: Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and β-lactamase

    Article Snippet: After 60 min, GEM particles and insoluble amylose azure were spun down, and the absorbance at 595 nm was determined. β-Lactamase activity was measured by adding 40 μl nitrocefin (CalBiochem) to GEM particles loaded with β-PA in 1 ml (final volume) of PBS.

    Techniques: Incubation

    Purified β-lactamase proteins. The six β-lactamase proteins were purified to > 90% homogeneity, as determined by SDS-PAGE. The values on the molecular weight ladder (MWL) are expressed in units of kilodaltons.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Binding Properties of a Peptide Derived from ?-Lactamase Inhibitory Protein

    doi: 10.1128/AAC.45.12.3279-3286.2001

    Figure Lengend Snippet: Purified β-lactamase proteins. The six β-lactamase proteins were purified to > 90% homogeneity, as determined by SDS-PAGE. The values on the molecular weight ladder (MWL) are expressed in units of kilodaltons.

    Article Snippet: The β-lactamase gene was induced by adding isopropyl-β- d -thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and further incubation at 25°C for 4 h. Following induction the cells were pelleted and the supernatant containing the secreted, soluble β-lactamase was concentrated to 100 ml with an Amicon Centriprep-10 concentrator (Millipore Corp.).

    Techniques: Purification, SDS Page, Molecular Weight

    ). Closed squares, protein kinase C peptide; open squares, cyclic BP46-51 peptide; closed circles, reduced BP46-51 peptide; open circles, BP41-50 peptide. Each datum point is the average for two independent experiments. (B) Inhibition assay of TEM-1 β-lactamase by wild-type BLIP. A K i of 0.23 nM was determined by using a nonlinear regression fit as described above.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Binding Properties of a Peptide Derived from ?-Lactamase Inhibitory Protein

    doi: 10.1128/AAC.45.12.3279-3286.2001

    Figure Lengend Snippet: ). Closed squares, protein kinase C peptide; open squares, cyclic BP46-51 peptide; closed circles, reduced BP46-51 peptide; open circles, BP41-50 peptide. Each datum point is the average for two independent experiments. (B) Inhibition assay of TEM-1 β-lactamase by wild-type BLIP. A K i of 0.23 nM was determined by using a nonlinear regression fit as described above.

    Article Snippet: The β-lactamase gene was induced by adding isopropyl-β- d -thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and further incubation at 25°C for 4 h. Following induction the cells were pelleted and the supernatant containing the secreted, soluble β-lactamase was concentrated to 100 ml with an Amicon Centriprep-10 concentrator (Millipore Corp.).

    Techniques: Inhibition, Transmission Electron Microscopy

    Inactivation of lasR confers increased β-lactamase activity and β-lactam tolerance. A. Ceftazidime-resistant colonies emerged in a lawn of CF416 or CF416L1 cells on LB agar with 20 µg ml −1 of ceftazidime (Petri dish upper and lower halves), but only the lasR mutant lawns yielded small partially resistant colonies whose growth is inhibited by the addition of 8 µg ml −1 of the β-lactamase inhibitor tazobactam (Petri dish lower halves). Photographs were taken after incubation overnight at 37°C followed by 3 days at 22°C. B. β-Lactamase activity in culture supernatants, measured as a change in optical density at 490 nm (ΔA 490 ) after addition of the chromogenic substrate nitrocefin. Cases with no change are marked with an asterisk. Values are the average of three technical replicates, and error bars show standard deviations. Equivalent results were obtained using a qualitative whole-culture assay. C. β-Lactamase activity in whole cultures of CF416 and CF416 lasR ::Gm. Values are the averages of three cultures, and error bars show standard error of the mean. One unit of β-lactamase is defined as 1 µmol of nitrocefin hydrolysed per min per mg of total protein. D. Reduced killing by ceftazidime of the lasR mutant CF416L1 relative to CF416 and CF416L1R. Values are the average cfu of two cultures, and error bars show standard deviations.

    Journal: Molecular microbiology

    Article Title: Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients

    doi: 10.1111/j.1365-2958.2007.05678.x

    Figure Lengend Snippet: Inactivation of lasR confers increased β-lactamase activity and β-lactam tolerance. A. Ceftazidime-resistant colonies emerged in a lawn of CF416 or CF416L1 cells on LB agar with 20 µg ml −1 of ceftazidime (Petri dish upper and lower halves), but only the lasR mutant lawns yielded small partially resistant colonies whose growth is inhibited by the addition of 8 µg ml −1 of the β-lactamase inhibitor tazobactam (Petri dish lower halves). Photographs were taken after incubation overnight at 37°C followed by 3 days at 22°C. B. β-Lactamase activity in culture supernatants, measured as a change in optical density at 490 nm (ΔA 490 ) after addition of the chromogenic substrate nitrocefin. Cases with no change are marked with an asterisk. Values are the average of three technical replicates, and error bars show standard deviations. Equivalent results were obtained using a qualitative whole-culture assay. C. β-Lactamase activity in whole cultures of CF416 and CF416 lasR ::Gm. Values are the averages of three cultures, and error bars show standard error of the mean. One unit of β-lactamase is defined as 1 µmol of nitrocefin hydrolysed per min per mg of total protein. D. Reduced killing by ceftazidime of the lasR mutant CF416L1 relative to CF416 and CF416L1R. Values are the average cfu of two cultures, and error bars show standard deviations.

    Article Snippet: To assay β-lactamase activity in culture supernatants, P. aeruginosa cultures grown overnight with LB broth were passed through a filter (0.22 µm pore size, Millipore), and the cell-free supernatants were assayed for β-lactamase activity 30 min after addition of nitrocefin (Calbiochem), as described by the manufacturer, except that A490 was measured, and corrected by subtracting the value measured for the supernatant without nitrocefin.

    Techniques: Activity Assay, Mutagenesis, Incubation

    Reporter assay for detection of AmpG permease activity. The β-lactamase assay was performed on Escherichia coli strains BW25113 (parental strain), TP73 (Δ ampD )/pNU305, AR74 (Δ ampG/ Δ ampD )/pNU305/ pACY-AR1 (empty vector), and AR74/pNU305/pAC-Tanf_08635. Activity was determined by chromogenic nitrocefin substrate induced at 0, 30, 60 min and absorbance was measured at OD 486 . Data are representative of three independent experiments with similar results. Each value represents the mean (±SD) of three values measured in one representative assay.

    Journal: Frontiers in Microbiology

    Article Title: Regulation and Molecular Basis of Environmental Muropeptide Uptake and Utilization in Fastidious Oral Anaerobe Tannerella forsythia

    doi: 10.3389/fmicb.2017.00648

    Figure Lengend Snippet: Reporter assay for detection of AmpG permease activity. The β-lactamase assay was performed on Escherichia coli strains BW25113 (parental strain), TP73 (Δ ampD )/pNU305, AR74 (Δ ampG/ Δ ampD )/pNU305/ pACY-AR1 (empty vector), and AR74/pNU305/pAC-Tanf_08635. Activity was determined by chromogenic nitrocefin substrate induced at 0, 30, 60 min and absorbance was measured at OD 486 . Data are representative of three independent experiments with similar results. Each value represents the mean (±SD) of three values measured in one representative assay.

    Article Snippet: Lactamase activity of each strain lysate was assayed using the chromogenic nitrocefin substrate (Calbiochem).

    Techniques: Reporter Assay, Activity Assay, Beta Lactamase Assay, Plasmid Preparation