p gingivalis Atcc Search Results


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  • 99
    Thermo Fisher rpmi 1640 medium
    Rpmi 1640 Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 75424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    ATCC p gingivalis atcc
    mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas <t>gingivalis</t> (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    P Gingivalis Atcc, supplied by ATCC, used in various techniques. Bioz Stars score: 78/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC p gingivalis atcc 33277
    mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas <t>gingivalis</t> (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    P Gingivalis Atcc 33277, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    ATCC culture p gingivalis atcc
    mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas <t>gingivalis</t> (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    Culture P Gingivalis Atcc, supplied by ATCC, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    ATCC rabbit anti p gingivalis atcc
    Fluorescence -microscopic analysis of co-localization between vacuolar- and cytosolic- P. <t>gingivalis</t> with NDP52 ubiquitin-binding-adaptor protein in primary GECs. (A) Primary GECs were infected with PgFbFP (green fluorescence) and after differential digitonin permeabilization stained with an anti- P. gingivalis antibody (Alexa 594, red fluorescence), followed by staining against NDP52 (Alexa 350, blue fluorescence). Cytoplasmic bacteria were detected as FbFP-and Alexa Fluor 594 positive (yellow) previously, whereas vacuolar bacteria were only FbFP-positive (green). 40x micrographs; Bar 10 µm. (B) Majority of cytosolic P. gingivalis was determined to show a significant association with NDP52 (red and blue = purple). Mean +/− SD, n > 3.
    Rabbit Anti P Gingivalis Atcc, supplied by ATCC, used in various techniques. Bioz Stars score: 87/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    InvivoGen p gingivalis
    Proliferation and differentiation of CD4 + T cells mediated by gingipains from P. <t>gingivalis</t> . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence/absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) or the isogenic gingipain-null mutant ΔKΔRAB for 6 h at MOI (multiplicity of infection) 1:50. Naive CD4 + T cells stained with CFSE were added and co-stimulated for 5 days. Proliferation of lymphocytes was measured at day 1, 3, and 5. (A) Completed results of proliferation on day 1, 3, and 5 as a percentage of CFSE low cells. Data are presented as a mean ± standard deviation of assays performed in triplicate using four independent donors, and were analyzed with a two way ANOVA with the Bonferoni's posttest correction (# P
    P Gingivalis, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    ATCC bacterial growth p gingivalis
    TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. <t>gingivalis</t> at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.
    Bacterial Growth P Gingivalis, supplied by ATCC, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    ATCC bacterial culture p gingivalis
    TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. <t>gingivalis</t> at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.
    Bacterial Culture P Gingivalis, supplied by ATCC, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    ATCC p gingivalis strain 381
    TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. <t>gingivalis</t> at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.
    P Gingivalis Strain 381, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ATCC p gingivalis w50 nidcr
    Comparative proteomic of particle-free SN from <t>W50-NIDCR</t> and W50-ATCC. ( A ) Various amounts of particle-free SN from W50-ATCC and W50-NIDCR were resolved by one-dimensional SDS-PAGE and protein bands were visualized by Silver staining. ( B ) Coomassie staining of the same SNs that had been concentrated to 10X as described indicated in the materials and methods. ( C ) Same as (A) except particle-free bacterial SN from W50-ATCC and W50-NIDCR were labeled green or red, respectively, and resolved by two-dimensional difference in-gel electrophoresis.
    P Gingivalis W50 Nidcr, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    ATCC porphyromonas gingivalis p gingivalis
    Comparative proteomic of particle-free SN from <t>W50-NIDCR</t> and W50-ATCC. ( A ) Various amounts of particle-free SN from W50-ATCC and W50-NIDCR were resolved by one-dimensional SDS-PAGE and protein bands were visualized by Silver staining. ( B ) Coomassie staining of the same SNs that had been concentrated to 10X as described indicated in the materials and methods. ( C ) Same as (A) except particle-free bacterial SN from W50-ATCC and W50-NIDCR were labeled green or red, respectively, and resolved by two-dimensional difference in-gel electrophoresis.
    Porphyromonas Gingivalis P Gingivalis, supplied by ATCC, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    ATCC p gingivalis strain 33277
    Comparative proteomic of particle-free SN from <t>W50-NIDCR</t> and W50-ATCC. ( A ) Various amounts of particle-free SN from W50-ATCC and W50-NIDCR were resolved by one-dimensional SDS-PAGE and protein bands were visualized by Silver staining. ( B ) Coomassie staining of the same SNs that had been concentrated to 10X as described indicated in the materials and methods. ( C ) Same as (A) except particle-free bacterial SN from W50-ATCC and W50-NIDCR were labeled green or red, respectively, and resolved by two-dimensional difference in-gel electrophoresis.
    P Gingivalis Strain 33277, supplied by ATCC, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    ATCC p gingivalis strain w50
    Comparative proteomic of particle-free SN from <t>W50-NIDCR</t> and W50-ATCC. ( A ) Various amounts of particle-free SN from W50-ATCC and W50-NIDCR were resolved by one-dimensional SDS-PAGE and protein bands were visualized by Silver staining. ( B ) Coomassie staining of the same SNs that had been concentrated to 10X as described indicated in the materials and methods. ( C ) Same as (A) except particle-free bacterial SN from W50-ATCC and W50-NIDCR were labeled green or red, respectively, and resolved by two-dimensional difference in-gel electrophoresis.
    P Gingivalis Strain W50, supplied by ATCC, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    70
    ATCC p gingivalis type strain
    Nearest neighbor ORF of IS 1126 from P. <t>gingivalis</t> ATCC 33277. The open bar represents the total flanking sequence, and the markers are in base pairs. IS 1126 is depicted as the inverted arrowhead, and its sequence (1,338 bp) has been deleted from the schematic. Homologous genes and ORFs are labeled and represented as solid arrows, which are drawn to scale. Also shown is the accession number of each homologous sequence.
    P Gingivalis Type Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 70/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    ATCC p gingivalis strain fdc381
    Nearest neighbor ORF of IS 1126 from P. <t>gingivalis</t> ATCC 33277. The open bar represents the total flanking sequence, and the markers are in base pairs. IS 1126 is depicted as the inverted arrowhead, and its sequence (1,338 bp) has been deleted from the schematic. Homologous genes and ORFs are labeled and represented as solid arrows, which are drawn to scale. Also shown is the accession number of each homologous sequence.
    P Gingivalis Strain Fdc381, supplied by ATCC, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ATCC p gingivalis jcm 8525
    Inhibition of planktonic growth of P. <t>gingivalis</t> by LF-related agents. About 10 4 cells/ml of P. gingivalis <t>JCM</t> 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P
    P Gingivalis Jcm 8525, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    ATCC p gingivalis groel expression vector p gingivalis genomic dna
    Inhibition of planktonic growth of P. <t>gingivalis</t> by LF-related agents. About 10 4 cells/ml of P. gingivalis <t>JCM</t> 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P
    P Gingivalis Groel Expression Vector P Gingivalis Genomic Dna, supplied by ATCC, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    ATCC p intermedia atcc
    Inhibition of planktonic growth of P. <t>gingivalis</t> by LF-related agents. About 10 4 cells/ml of P. gingivalis <t>JCM</t> 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P
    P Intermedia Atcc, supplied by ATCC, used in various techniques. Bioz Stars score: 82/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    ATCC p gingivalis w83 dna
    Inhibition of planktonic growth of P. <t>gingivalis</t> by LF-related agents. About 10 4 cells/ml of P. gingivalis <t>JCM</t> 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P
    P Gingivalis W83 Dna, supplied by ATCC, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    ATCC microbial inocula p gingivalis fdc 381
    Inhibition of planktonic growth of P. <t>gingivalis</t> by LF-related agents. About 10 4 cells/ml of P. gingivalis <t>JCM</t> 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P
    Microbial Inocula P Gingivalis Fdc 381, supplied by ATCC, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    ATCC antibacterial activity against p gingivalis
    Inhibition of planktonic growth of P. <t>gingivalis</t> by LF-related agents. About 10 4 cells/ml of P. gingivalis <t>JCM</t> 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P
    Antibacterial Activity Against P Gingivalis, supplied by ATCC, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    ATCC bacterial cell culture p gingivalis
    Inhibition of planktonic growth of P. <t>gingivalis</t> by LF-related agents. About 10 4 cells/ml of P. gingivalis <t>JCM</t> 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P
    Bacterial Cell Culture P Gingivalis, supplied by ATCC, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    ATCC macrophage preparation p gingivalis 381
    Inhibition of planktonic growth of P. <t>gingivalis</t> by LF-related agents. About 10 4 cells/ml of P. gingivalis <t>JCM</t> 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P
    Macrophage Preparation P Gingivalis 381, supplied by ATCC, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    ATCC p gingivalis wild type strain
    Sensitivity of P. <t>gingivalis</t> mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).
    P Gingivalis Wild Type Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    ATCC divergent strains p gingivalis 33277
    Sensitivity of P. <t>gingivalis</t> mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).
    Divergent Strains P Gingivalis 33277, supplied by ATCC, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    ATCC bacterial growth conditions p gingivalis w83
    Sensitivity of P. <t>gingivalis</t> mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).
    Bacterial Growth Conditions P Gingivalis W83, supplied by ATCC, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC s mutans atcc 25175
    Sensitivity of P. <t>gingivalis</t> mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).
    S Mutans Atcc 25175, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    ATCC bacteria culture p gingivalis 381
    Sensitivity of P. <t>gingivalis</t> mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).
    Bacteria Culture P Gingivalis 381, supplied by ATCC, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    ATCC gram negative bacteria p gingivalis w50
    Sensitivity of P. <t>gingivalis</t> mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).
    Gram Negative Bacteria P Gingivalis W50, supplied by ATCC, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    ATCC culture conditions wild type p gingivalis
    Cleavage (A) and activation (B) of procaspase-3 by P. <t>gingivalis</t> -excreted gingipains. (A) Incubation of pure procaspase-3 with caspase-8 (4 or 8 ng) or culture supernatants ('sup') of wild type (WT) or triple gingipain mutant (MT) P. gingivalis (20 or 40 ng protein content, labeled over lanes) were done as described under Experimental Procedures. The products were analyzed by SDS-PAGE and silver-staining. The full-length procaspase-3 (34 kDa) and the processed active fragments (17 and 12 kDa) are marked by open and closed arrowheads, respectively. (B) Activity of the processed caspase-3 generated in Panel A was measured using a colorimetric peptide substrate described under Experimental Procedures. The enzymes used to cleave procsapase-3 are: 20 ng of wild type P. gingivalis sup (Triangle); 4 ng caspase-8 (Squares); 20 ng of gingipain mutant P. gingivalis sup (Open circles); 20 ng of wild type P. gingivalis sup, preheated at 65°C for 15 min (Diamonds); none (Closed circles). The error bars are derived from three independent experiments.
    Culture Conditions Wild Type P Gingivalis, supplied by ATCC, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    ATCC bacterial culture p gingivalis strain
    Cleavage (A) and activation (B) of procaspase-3 by P. <t>gingivalis</t> -excreted gingipains. (A) Incubation of pure procaspase-3 with caspase-8 (4 or 8 ng) or culture supernatants ('sup') of wild type (WT) or triple gingipain mutant (MT) P. gingivalis (20 or 40 ng protein content, labeled over lanes) were done as described under Experimental Procedures. The products were analyzed by SDS-PAGE and silver-staining. The full-length procaspase-3 (34 kDa) and the processed active fragments (17 and 12 kDa) are marked by open and closed arrowheads, respectively. (B) Activity of the processed caspase-3 generated in Panel A was measured using a colorimetric peptide substrate described under Experimental Procedures. The enzymes used to cleave procsapase-3 are: 20 ng of wild type P. gingivalis sup (Triangle); 4 ng caspase-8 (Squares); 20 ng of gingipain mutant P. gingivalis sup (Open circles); 20 ng of wild type P. gingivalis sup, preheated at 65°C for 15 min (Diamonds); none (Closed circles). The error bars are derived from three independent experiments.
    Bacterial Culture P Gingivalis Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    ATCC western blot p gingivalis strains w83
    Cleavage (A) and activation (B) of procaspase-3 by P. <t>gingivalis</t> -excreted gingipains. (A) Incubation of pure procaspase-3 with caspase-8 (4 or 8 ng) or culture supernatants ('sup') of wild type (WT) or triple gingipain mutant (MT) P. gingivalis (20 or 40 ng protein content, labeled over lanes) were done as described under Experimental Procedures. The products were analyzed by SDS-PAGE and silver-staining. The full-length procaspase-3 (34 kDa) and the processed active fragments (17 and 12 kDa) are marked by open and closed arrowheads, respectively. (B) Activity of the processed caspase-3 generated in Panel A was measured using a colorimetric peptide substrate described under Experimental Procedures. The enzymes used to cleave procsapase-3 are: 20 ng of wild type P. gingivalis sup (Triangle); 4 ng caspase-8 (Squares); 20 ng of gingipain mutant P. gingivalis sup (Open circles); 20 ng of wild type P. gingivalis sup, preheated at 65°C for 15 min (Diamonds); none (Closed circles). The error bars are derived from three independent experiments.
    Western Blot P Gingivalis Strains W83, supplied by ATCC, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter.

    Techniques: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    mRNA expression of innate immune markers CC chemokine ligand 20 (CCL20) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of ( b ) CCL20 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of CCL20 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: mRNA expression of innate immune markers CC chemokine ligand 20 (CCL20) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of ( b ) CCL20 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of CCL20 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter.

    Techniques: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    Differential decreased mRNA expression of HDAC1, HDAC2 and DNMT1 in gingival epithelial cells in response to various doses of oral bacteria. mRNA expression of ( a ) DNA methyltransferase (DNMT1), ( b ) histone deacetylase 1 (HDAC1), and ( c ) histone deacetylase 2 (HDAC2) are differentially decreased in gingival epithelial cells (GECs) in response to various doses of Porphyromonas gingivalis vs. Fusobacterium nucleatum . GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, 100:1, and 200:1 for 24 h. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the unstimulated control after normalization with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH ). The data are derived from three different cell donors tested in duplicate. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Differential decreased mRNA expression of HDAC1, HDAC2 and DNMT1 in gingival epithelial cells in response to various doses of oral bacteria. mRNA expression of ( a ) DNA methyltransferase (DNMT1), ( b ) histone deacetylase 1 (HDAC1), and ( c ) histone deacetylase 2 (HDAC2) are differentially decreased in gingival epithelial cells (GECs) in response to various doses of Porphyromonas gingivalis vs. Fusobacterium nucleatum . GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, 100:1, and 200:1 for 24 h. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the unstimulated control after normalization with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH ). The data are derived from three different cell donors tested in duplicate. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter.

    Techniques: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, CTL Assay

    Protein levels of histone H3 methylated at Lys4 were evaluated with PathScan enzyme-linked immunosorbent assay (ELISA). Gingival epithelial cells (GECs) were exposed to Porphyromonas gingivalis or Fusobacterium nucleatum for 24 h at multiplicity of infection (MOI) of 100:1, and then nuclear protein was extracted followed by sonication. The H3 tri-methylated at Lys4 was captured by coated antibody after incubation with cell lysates, and histone H3 protein level was quantified according to the absorbance readings at 450 nm. Protein expression was expressed as the ratio of absorbance readings normalized to relative protein amount. The data are average from three different donor cells tested with standard error deviation. The asterisks indicate the significant difference vs. the respective unstimulated control (** P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Protein levels of histone H3 methylated at Lys4 were evaluated with PathScan enzyme-linked immunosorbent assay (ELISA). Gingival epithelial cells (GECs) were exposed to Porphyromonas gingivalis or Fusobacterium nucleatum for 24 h at multiplicity of infection (MOI) of 100:1, and then nuclear protein was extracted followed by sonication. The H3 tri-methylated at Lys4 was captured by coated antibody after incubation with cell lysates, and histone H3 protein level was quantified according to the absorbance readings at 450 nm. Protein expression was expressed as the ratio of absorbance readings normalized to relative protein amount. The data are average from three different donor cells tested with standard error deviation. The asterisks indicate the significant difference vs. the respective unstimulated control (** P

    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter.

    Techniques: Methylation, Enzyme-linked Immunosorbent Assay, Infection, Sonication, Incubation, Expressing

    Protein levels of histone deacetylases 1 and 2 (HDAC1 and HDAC2) and DNA methyltransferase (DNMT1) are differentially expressed in gingival epithelial cells (GECs) in response to Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn). GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 100:1 for 24 h. Nuclear proteins were extracted, denatured at 70 °C for 10 min, and separated by NuPAGE electrophoresis system. Nuclear extracts of Hela cells probed with individual primary antibody were used as positive controls. The data are derived from two different cell donors tested in duplicate.

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Protein levels of histone deacetylases 1 and 2 (HDAC1 and HDAC2) and DNA methyltransferase (DNMT1) are differentially expressed in gingival epithelial cells (GECs) in response to Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn). GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 100:1 for 24 h. Nuclear proteins were extracted, denatured at 70 °C for 10 min, and separated by NuPAGE electrophoresis system. Nuclear extracts of Hela cells probed with individual primary antibody were used as positive controls. The data are derived from two different cell donors tested in duplicate.

    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter.

    Techniques: Infection, Electrophoresis, Derivative Assay

    Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of ( a ) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and ( b ) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum . Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of ( a ) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and ( b ) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum . Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    Interleukin-8 (IL-8) secretion is increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Secretion of IL-8 in response to various oral bacteria is evaluated by enzyme-linked immunosorbent assay (ELISA). Cell-free supernatant was collected and the amount of IL-8 secreted is shown in pg ml –1 . Unstimulated cells (UN) are used as controls in each experiment. Data from duplicates with cells from three different donors are shown. No significant changes were found in the secretion of IL-8 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Interleukin-8 (IL-8) secretion is increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Secretion of IL-8 in response to various oral bacteria is evaluated by enzyme-linked immunosorbent assay (ELISA). Cell-free supernatant was collected and the amount of IL-8 secreted is shown in pg ml –1 . Unstimulated cells (UN) are used as controls in each experiment. Data from duplicates with cells from three different donors are shown. No significant changes were found in the secretion of IL-8 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter.

    Techniques: Histone Deacetylase Assay, Infection, Enzyme-linked Immunosorbent Assay, CTL Assay

    Distribution of the dead/viable bacteria in the biofilms formed by a P. intermedia and P. gingivalis co-culture observed by confocal laser scanning microscopy. Acridine orange (AO) and ethidium bromide (EB) staining; magnification, ×200. (A) Intracellular proteins of S. sanguinis ; (B) exocrine proteins of S. sanguinis ; and (C) deionized water. P. intermedia, Prevotella intermedia; P. gingivalis, Porphyromonas gingivalis; S. sanguinis, Streptococcus sanguinis.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Antagonistic effect of protein extracts from Streptococcus sanguinis on pathogenic bacteria and fungi of the oral cavity

    doi: 10.3892/etm.2014.1618

    Figure Lengend Snippet: Distribution of the dead/viable bacteria in the biofilms formed by a P. intermedia and P. gingivalis co-culture observed by confocal laser scanning microscopy. Acridine orange (AO) and ethidium bromide (EB) staining; magnification, ×200. (A) Intracellular proteins of S. sanguinis ; (B) exocrine proteins of S. sanguinis ; and (C) deionized water. P. intermedia, Prevotella intermedia; P. gingivalis, Porphyromonas gingivalis; S. sanguinis, Streptococcus sanguinis.

    Article Snippet: P. intermedia and P. gingivalis The standard strains ATCC 25611 and ATCC 33277 of P. intermedia and P. gingivalis , respectively, were purchased from Beijing Stomatological Hospital, Capital Medical University (Beijing, China).

    Techniques: Co-Culture Assay, Confocal Laser Scanning Microscopy, Staining

    Evidence for allelic replacement of prtP in P. gingivalis W12 by a double-crossover event. Digested fragments of P. gingivalis genomic DNA were detected with a probe prepared from the 2.5-kb fragment of pMJF3 containing tetA (Q) (A) or a 1.16-kb Hin dIII fragment from pNH5 containing DNA absent in pML1 (B). (A) Lanes: 1, genomic DNA from strain W12 digested with Bam HI; 2 to 6, genomic DNA from strain W12 prtP :: tetA (Q) digested with Ban I (lane 2), Ahd I plus Pvu II (lane 3), Ahd I (lane 4), Bam HI plus Pvu II (lane 5), or Bam HI (lane 6). (B) Lanes 1 and 2, Bam HI-digested genomic DNA from strain W12 (lane 1) or strain W12 prtP :: tetA (Q) (lane 2). The sizes (in kilobases) and locations of DNA standards are indicated.

    Journal: Journal of Bacteriology

    Article Title: Activities of the Porphyromonas gingivalis PrtP Proteinase Determined by Construction of prtP-Deficient Mutants and Expression of the Gene in Bacteroides Species

    doi:

    Figure Lengend Snippet: Evidence for allelic replacement of prtP in P. gingivalis W12 by a double-crossover event. Digested fragments of P. gingivalis genomic DNA were detected with a probe prepared from the 2.5-kb fragment of pMJF3 containing tetA (Q) (A) or a 1.16-kb Hin dIII fragment from pNH5 containing DNA absent in pML1 (B). (A) Lanes: 1, genomic DNA from strain W12 digested with Bam HI; 2 to 6, genomic DNA from strain W12 prtP :: tetA (Q) digested with Ban I (lane 2), Ahd I plus Pvu II (lane 3), Ahd I (lane 4), Bam HI plus Pvu II (lane 5), or Bam HI (lane 6). (B) Lanes 1 and 2, Bam HI-digested genomic DNA from strain W12 (lane 1) or strain W12 prtP :: tetA (Q) (lane 2). The sizes (in kilobases) and locations of DNA standards are indicated.

    Article Snippet: Furthermore, P. gingivalis W12, ATCC 33277, KDP110, and HG66 with knockout mutations in prtP were constructed by allelic replacement.

    Techniques:

    Alteration of the transcriptional landscape of TIGKs infected with P. gingivalis in the presence of absence of active PPAD. Significant changes in GS, e.g ., biological processes and pathways, induced in TIGKs upon infection with PPAD-competent P. gingivalis were mapped. The fill color of each node represents the direction and significance level for this comparison (orange, enrichment, FDR q

    Journal: Scientific Reports

    Article Title: Impact of Porphyromonas gingivalis Peptidylarginine Deiminase on Bacterial Biofilm Formation, Epithelial Cell Invasion, and Epithelial Cell Transcriptional Landscape

    doi: 10.1038/s41598-018-32603-y

    Figure Lengend Snippet: Alteration of the transcriptional landscape of TIGKs infected with P. gingivalis in the presence of absence of active PPAD. Significant changes in GS, e.g ., biological processes and pathways, induced in TIGKs upon infection with PPAD-competent P. gingivalis were mapped. The fill color of each node represents the direction and significance level for this comparison (orange, enrichment, FDR q

    Article Snippet: Global gene expression profiles were generated from the following samples using HumanHT-12 v4 Expression BeadChip kits (Illumina, San Diego, USA): (i) uninfected TIGKs; (ii) TIGKs infected with P. gingivalis WT (ATCC 33277); and (iii) TIGKS infected with P. gingivalis PPADC351A .

    Techniques: Infection

    Identification and functional annotation of TIGK genes significantly dependent on PPAD activity. ( A ) Scatterplot of log2 fold-change (log2FC) in gene expression of TIGKs infected with P. gingivalis WT vs. control (y-axis), and P. gingivalis PPAD C351A vs. P. gingivalis WT (x-axis). Genes altered exclusively in the P. gingivalis WT vs. control comparison are displayed in red when significantly up-regulated (log2FC > 0.5, FDR q

    Journal: Scientific Reports

    Article Title: Impact of Porphyromonas gingivalis Peptidylarginine Deiminase on Bacterial Biofilm Formation, Epithelial Cell Invasion, and Epithelial Cell Transcriptional Landscape

    doi: 10.1038/s41598-018-32603-y

    Figure Lengend Snippet: Identification and functional annotation of TIGK genes significantly dependent on PPAD activity. ( A ) Scatterplot of log2 fold-change (log2FC) in gene expression of TIGKs infected with P. gingivalis WT vs. control (y-axis), and P. gingivalis PPAD C351A vs. P. gingivalis WT (x-axis). Genes altered exclusively in the P. gingivalis WT vs. control comparison are displayed in red when significantly up-regulated (log2FC > 0.5, FDR q

    Article Snippet: Global gene expression profiles were generated from the following samples using HumanHT-12 v4 Expression BeadChip kits (Illumina, San Diego, USA): (i) uninfected TIGKs; (ii) TIGKs infected with P. gingivalis WT (ATCC 33277); and (iii) TIGKS infected with P. gingivalis PPADC351A .

    Techniques: Functional Assay, Activity Assay, Expressing, Infection

    The effect of P. gingivalis PPAD on bacterial abundance ( a ), species composition ( b ) and ( c ) the biofilm structure in multispecies biofilms. A biofilm consisting of T. forsythia , F. nucleatum , A. naeslundii , S. gordonii , and one of three P. gingivalis strains, wild type (WT), ppad deletion strain ( Δppad ), or a strain harboring inactivated PPAD (C351A), was cultured for 48 h, following which ( a,b ) the bacterial DNA was extracted and assessed via qPCR, ( c ) biofilm was fixed and observed via SEM under 1500x magnification. The ( a ) bacterial load and ( b ) species composition of each biofilm model are presented as mean ± SD from three independent experiments. Data were plotted on a logarithmic scale.

    Journal: Scientific Reports

    Article Title: Impact of Porphyromonas gingivalis Peptidylarginine Deiminase on Bacterial Biofilm Formation, Epithelial Cell Invasion, and Epithelial Cell Transcriptional Landscape

    doi: 10.1038/s41598-018-32603-y

    Figure Lengend Snippet: The effect of P. gingivalis PPAD on bacterial abundance ( a ), species composition ( b ) and ( c ) the biofilm structure in multispecies biofilms. A biofilm consisting of T. forsythia , F. nucleatum , A. naeslundii , S. gordonii , and one of three P. gingivalis strains, wild type (WT), ppad deletion strain ( Δppad ), or a strain harboring inactivated PPAD (C351A), was cultured for 48 h, following which ( a,b ) the bacterial DNA was extracted and assessed via qPCR, ( c ) biofilm was fixed and observed via SEM under 1500x magnification. The ( a ) bacterial load and ( b ) species composition of each biofilm model are presented as mean ± SD from three independent experiments. Data were plotted on a logarithmic scale.

    Article Snippet: Global gene expression profiles were generated from the following samples using HumanHT-12 v4 Expression BeadChip kits (Illumina, San Diego, USA): (i) uninfected TIGKs; (ii) TIGKs infected with P. gingivalis WT (ATCC 33277); and (iii) TIGKS infected with P. gingivalis PPADC351A .

    Techniques: Cell Culture, Real-time Polymerase Chain Reaction

    P. gingivalis invasion of human oral keratinocytes. CTV-labeled TIGKs were infected with various CFSE-labeled strains of P. gingivalis (MOI = 200) for 90 min. Metronidazole was added for an additional 1 h, following which the cells were fixed and analyzed by flow cytometry. Cells were first gated on the basis of a forward scatter/side scatter (FSC-A/SSC-A) plot ( a ). The events were then visualized using FSC-A/FSC-H dot plot, and single cells were gated ( b ). TIGKs were identified on the basis of CTV positivity ( c ). TIGKs invaded by P. gingivalis were subsequently defined as the CFSE + cells within the CTV + keratinocyte population ( d ). Overall, 20,000 events were analyzed, and the results are presented as the percentage of TIGKs infected with P. gingivalis wild-type ( e ), Δppad ( f ), and C351A ( g ).

    Journal: Scientific Reports

    Article Title: Impact of Porphyromonas gingivalis Peptidylarginine Deiminase on Bacterial Biofilm Formation, Epithelial Cell Invasion, and Epithelial Cell Transcriptional Landscape

    doi: 10.1038/s41598-018-32603-y

    Figure Lengend Snippet: P. gingivalis invasion of human oral keratinocytes. CTV-labeled TIGKs were infected with various CFSE-labeled strains of P. gingivalis (MOI = 200) for 90 min. Metronidazole was added for an additional 1 h, following which the cells were fixed and analyzed by flow cytometry. Cells were first gated on the basis of a forward scatter/side scatter (FSC-A/SSC-A) plot ( a ). The events were then visualized using FSC-A/FSC-H dot plot, and single cells were gated ( b ). TIGKs were identified on the basis of CTV positivity ( c ). TIGKs invaded by P. gingivalis were subsequently defined as the CFSE + cells within the CTV + keratinocyte population ( d ). Overall, 20,000 events were analyzed, and the results are presented as the percentage of TIGKs infected with P. gingivalis wild-type ( e ), Δppad ( f ), and C351A ( g ).

    Article Snippet: Global gene expression profiles were generated from the following samples using HumanHT-12 v4 Expression BeadChip kits (Illumina, San Diego, USA): (i) uninfected TIGKs; (ii) TIGKs infected with P. gingivalis WT (ATCC 33277); and (iii) TIGKS infected with P. gingivalis PPADC351A .

    Techniques: Labeling, Infection, Flow Cytometry, Cytometry

    IL-8 secretion from HOK-18A following sequential challenge of F. nucleatum and P. gingivalis . (○) Control group without bacteria. Supernatants were collected at 18 h for IL-8 ELISA. (■) F. nucleatum (MOI, 300:1) was added to cell monolayers and supernatants were collected for IL-8 measurement at the 4-, 8-, or 18-h time point. ↓, P. gingivalis 381 (MOI, 500:1) was added to cell monolayers 4 h (▵) or 8 h (⧫) after the addition of F. nucleatum (MOI, 300:1), and the supernatant was collected at 18 h.

    Journal: Infection and Immunity

    Article Title: Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects of Porphyromonas gingivalis via Antagonistic Mechanisms

    doi: 10.1128/IAI.69.3.1364-1372.2001

    Figure Lengend Snippet: IL-8 secretion from HOK-18A following sequential challenge of F. nucleatum and P. gingivalis . (○) Control group without bacteria. Supernatants were collected at 18 h for IL-8 ELISA. (■) F. nucleatum (MOI, 300:1) was added to cell monolayers and supernatants were collected for IL-8 measurement at the 4-, 8-, or 18-h time point. ↓, P. gingivalis 381 (MOI, 500:1) was added to cell monolayers 4 h (▵) or 8 h (⧫) after the addition of F. nucleatum (MOI, 300:1), and the supernatant was collected at 18 h.

    Article Snippet: Thus, P. gingivalis 381, ATCC 22377, and W50 up-regulated IL-8 and ICAM-1 mRNA when the epithelial-cell cultures were washed at the 2-h time point and down-regulated IL-8 below the baseline level when the bacteria were continuously cocultured with the epithelial cells.

    Techniques: Enzyme-linked Immunosorbent Assay

    IL-8 and ICAM-1 mRNA levels in gingival epithelial cells following bacterial challenge. Confluent HOK-18A cell monolayers were infected with  F. nucleatum  or various  P. gingivalis  strains at a MOI of 500:1. The bacterium–epithelial-cell coculture period was either 2, 4, or 6 h. RNA was isolated at 4 or 6 h after infection, and 8 to 12 μg of total RNA was subjected to Northern blot analysis. For the 2-h coculture group, the culture was washed and fresh medium containing appropriate antibiotics was added at 2 h and the cultures were further incubated for another 2 to 4 h before harvesting. For the 4- or 6-h coculture group, the bacteria were continuously cocultured with the epithelial cells to 4 or 6 h (see diagram in the bottom panel). (A) Epithelial cells were challenged with  F. nucleatum  12230,  P. gingivalis  381, or  P. gingivalis  W50 for 2 h (washed) or 6 h (continuous), and RNA was isolated at 6 h. (B and C) Epithelial cells were challenged with  P. gingivalis  33277 (B) or  F. nucleatum  12230 (C) for 2 h (washed) or 4 h (continuous) and RNA was isolated at 4 h. The levels of mRNA from the Northern blot analysis (shown in the top panel) are plotted and shown in the middle panel. Mock, no bacteria were added to the cultures.  Fn ,  F. nucleatum ;  Pg ,  P. gingivalis ;  W , 2-h coculture group that was washed at the 2-h time point;  C , 4- or 6-h coculture group, bacteria continuously cocultured with the epithelial cells.

    Journal: Infection and Immunity

    Article Title: Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects of Porphyromonas gingivalis via Antagonistic Mechanisms

    doi: 10.1128/IAI.69.3.1364-1372.2001

    Figure Lengend Snippet: IL-8 and ICAM-1 mRNA levels in gingival epithelial cells following bacterial challenge. Confluent HOK-18A cell monolayers were infected with F. nucleatum or various P. gingivalis strains at a MOI of 500:1. The bacterium–epithelial-cell coculture period was either 2, 4, or 6 h. RNA was isolated at 4 or 6 h after infection, and 8 to 12 μg of total RNA was subjected to Northern blot analysis. For the 2-h coculture group, the culture was washed and fresh medium containing appropriate antibiotics was added at 2 h and the cultures were further incubated for another 2 to 4 h before harvesting. For the 4- or 6-h coculture group, the bacteria were continuously cocultured with the epithelial cells to 4 or 6 h (see diagram in the bottom panel). (A) Epithelial cells were challenged with F. nucleatum 12230, P. gingivalis 381, or P. gingivalis W50 for 2 h (washed) or 6 h (continuous), and RNA was isolated at 6 h. (B and C) Epithelial cells were challenged with P. gingivalis 33277 (B) or F. nucleatum 12230 (C) for 2 h (washed) or 4 h (continuous) and RNA was isolated at 4 h. The levels of mRNA from the Northern blot analysis (shown in the top panel) are plotted and shown in the middle panel. Mock, no bacteria were added to the cultures. Fn , F. nucleatum ; Pg , P. gingivalis ; W , 2-h coculture group that was washed at the 2-h time point; C , 4- or 6-h coculture group, bacteria continuously cocultured with the epithelial cells.

    Article Snippet: Thus, P. gingivalis 381, ATCC 22377, and W50 up-regulated IL-8 and ICAM-1 mRNA when the epithelial-cell cultures were washed at the 2-h time point and down-regulated IL-8 below the baseline level when the bacteria were continuously cocultured with the epithelial cells.

    Techniques: Infection, Isolation, Northern Blot, Incubation

    Fas expression by CD3 + T cells derived from chronic periodontitis (CP) patients and subjects without periodontitis (NP) upon stimulation (48 h) with P. gingivalis ATCC 33277 crude extract (Pg33277), purified recombinant P. gingivalis HmuY protein (HmuY), or without stimulus (Cells) as evaluated by flow cytometry.

    Journal: BMC Microbiology

    Article Title: Porphyromonas gingivalis HmuY stimulates expression of Bcl-2 and Fas by human CD3+ T cells

    doi: 10.1186/1471-2180-13-206

    Figure Lengend Snippet: Fas expression by CD3 + T cells derived from chronic periodontitis (CP) patients and subjects without periodontitis (NP) upon stimulation (48 h) with P. gingivalis ATCC 33277 crude extract (Pg33277), purified recombinant P. gingivalis HmuY protein (HmuY), or without stimulus (Cells) as evaluated by flow cytometry.

    Article Snippet: Cells were also incubated with 5 μg/mL of pokeweed mitogen (PWM) as a positive control, 0.5 μg/mL of P. gingivalis extract (ATCC 33277), 2.5 μg/mL of HmuY, or in the absence of antigens (Cells).

    Techniques: Expressing, Derivative Assay, Purification, Recombinant, Flow Cytometry, Cytometry

    Bcl-2 expression by CD3 + T cells derived from chronic periodontitis (CP) patients and subjects without periodontitis (NP) upon stimulation (48 h) with P. gingivalis ATCC 33277 crude extract (Pg33277), purified recombinant P. gingivalis HmuY protein (HmuY), or without stimulus (Cells) as evaluated by flow cytometry. * p = 0.043, ‡ p = 0,011.

    Journal: BMC Microbiology

    Article Title: Porphyromonas gingivalis HmuY stimulates expression of Bcl-2 and Fas by human CD3+ T cells

    doi: 10.1186/1471-2180-13-206

    Figure Lengend Snippet: Bcl-2 expression by CD3 + T cells derived from chronic periodontitis (CP) patients and subjects without periodontitis (NP) upon stimulation (48 h) with P. gingivalis ATCC 33277 crude extract (Pg33277), purified recombinant P. gingivalis HmuY protein (HmuY), or without stimulus (Cells) as evaluated by flow cytometry. * p = 0.043, ‡ p = 0,011.

    Article Snippet: Cells were also incubated with 5 μg/mL of pokeweed mitogen (PWM) as a positive control, 0.5 μg/mL of P. gingivalis extract (ATCC 33277), 2.5 μg/mL of HmuY, or in the absence of antigens (Cells).

    Techniques: Expressing, Derivative Assay, Purification, Recombinant, Flow Cytometry, Cytometry

    IRAK1 expression was inhibited by miR-146a in B cells after challenged with P. gingivalis LPS miR-146a mimic could reduce the mRNA and protein levels of IRAK1 as detected by PCR (A) and ELISA (B). Also, addition of miR-146a inhibited TRAF6 expression in B cells at both mRNA (C) and protein level (D). Inhibition of miR-146a by its inhibitor significantly elevated both mRNA and protein levels of IRAK1 expression in B cells (E and F), as well as mRNA (G) and protein (H) levels of TRAF6 expression in B cells. The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. One-way ANOVA. * p

    Journal: Biochimica et biophysica acta

    Article Title: miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

    doi: 10.1016/j.bbadis.2017.12.035

    Figure Lengend Snippet: IRAK1 expression was inhibited by miR-146a in B cells after challenged with P. gingivalis LPS miR-146a mimic could reduce the mRNA and protein levels of IRAK1 as detected by PCR (A) and ELISA (B). Also, addition of miR-146a inhibited TRAF6 expression in B cells at both mRNA (C) and protein level (D). Inhibition of miR-146a by its inhibitor significantly elevated both mRNA and protein levels of IRAK1 expression in B cells (E and F), as well as mRNA (G) and protein (H) levels of TRAF6 expression in B cells. The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. One-way ANOVA. * p

    Article Snippet: For groups 2–4, mice were orally infected with live P. gingivalis bacteria (ATCC 33277) pre-mixed with an equal volume of sterile 2% (wt/vol) low-viscosity carboxymethylcellulose (CMC).

    Techniques: Expressing, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Inhibition

    The inflammatory responses in B cells were activated by P. gingivalis LPS After P. gingivalis LPS treatment, mRNA levels of inflammatory cytokines including IL-1β (A), IL-6 (B) and IL-10 (C) were significantly increased in B cells. IRAK1 mRNA level was also increased (D). However, TRAF6 mRNA level was not changed (E). The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. Student’s t-test. * p

    Journal: Biochimica et biophysica acta

    Article Title: miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

    doi: 10.1016/j.bbadis.2017.12.035

    Figure Lengend Snippet: The inflammatory responses in B cells were activated by P. gingivalis LPS After P. gingivalis LPS treatment, mRNA levels of inflammatory cytokines including IL-1β (A), IL-6 (B) and IL-10 (C) were significantly increased in B cells. IRAK1 mRNA level was also increased (D). However, TRAF6 mRNA level was not changed (E). The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. Student’s t-test. * p

    Article Snippet: For groups 2–4, mice were orally infected with live P. gingivalis bacteria (ATCC 33277) pre-mixed with an equal volume of sterile 2% (wt/vol) low-viscosity carboxymethylcellulose (CMC).

    Techniques:

    The expression of miR-146a in B cells was induced by P. gingivalis LPS The expression of miR-146a in B cells was detected by qRT-PCR after P. gingivalis LPS-stimulation. The miR-146a expression was increased at both 24 h and 48 h. The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. Student’s t-test. * p

    Journal: Biochimica et biophysica acta

    Article Title: miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

    doi: 10.1016/j.bbadis.2017.12.035

    Figure Lengend Snippet: The expression of miR-146a in B cells was induced by P. gingivalis LPS The expression of miR-146a in B cells was detected by qRT-PCR after P. gingivalis LPS-stimulation. The miR-146a expression was increased at both 24 h and 48 h. The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. Student’s t-test. * p

    Article Snippet: For groups 2–4, mice were orally infected with live P. gingivalis bacteria (ATCC 33277) pre-mixed with an equal volume of sterile 2% (wt/vol) low-viscosity carboxymethylcellulose (CMC).

    Techniques: Expressing, Quantitative RT-PCR

    The mRNA expressions of inflammatory cytokines were inhibited by miR-146a in B cells after challenged with P. gingivalis LPS After overexpression of miR-146a in B cells, mRNA levels of IL-1β (A), IL-6 (B) and IL-10 (C) were significantly decreased. However, after blockade of miR-146a by its inhibitor, the mRNA levels of IL-1β (D), IL-6 (E) and IL-10 (F) were significantly increased. The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. One-way ANOVA. * p

    Journal: Biochimica et biophysica acta

    Article Title: miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

    doi: 10.1016/j.bbadis.2017.12.035

    Figure Lengend Snippet: The mRNA expressions of inflammatory cytokines were inhibited by miR-146a in B cells after challenged with P. gingivalis LPS After overexpression of miR-146a in B cells, mRNA levels of IL-1β (A), IL-6 (B) and IL-10 (C) were significantly decreased. However, after blockade of miR-146a by its inhibitor, the mRNA levels of IL-1β (D), IL-6 (E) and IL-10 (F) were significantly increased. The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. One-way ANOVA. * p

    Article Snippet: For groups 2–4, mice were orally infected with live P. gingivalis bacteria (ATCC 33277) pre-mixed with an equal volume of sterile 2% (wt/vol) low-viscosity carboxymethylcellulose (CMC).

    Techniques: Over Expression

    miR-146a inhibited inflammatory cytokine secretion from in B cells after challenged with P. gingivalis LPS The secretions of IL-1β (A), IL-6 (B) and IL-10 (C) decreased in presence of miR-146a mimic. The productions of IL-1β (D), IL-6 (E) and IL-10 (F) were enhanced by miR-146a inhibitor. The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. One-way ANOVA. * p

    Journal: Biochimica et biophysica acta

    Article Title: miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

    doi: 10.1016/j.bbadis.2017.12.035

    Figure Lengend Snippet: miR-146a inhibited inflammatory cytokine secretion from in B cells after challenged with P. gingivalis LPS The secretions of IL-1β (A), IL-6 (B) and IL-10 (C) decreased in presence of miR-146a mimic. The productions of IL-1β (D), IL-6 (E) and IL-10 (F) were enhanced by miR-146a inhibitor. The experiments were performed in triplicate. n=6–8. Data are presented as mean±SD. One-way ANOVA. * p

    Article Snippet: For groups 2–4, mice were orally infected with live P. gingivalis bacteria (ATCC 33277) pre-mixed with an equal volume of sterile 2% (wt/vol) low-viscosity carboxymethylcellulose (CMC).

    Techniques:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Journal: Infection and Immunity

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    doi:

    Figure Lengend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Article Snippet: Under high-stringency conditions, this probe hybridized with DNA of all P. gingivalis strains (ATCC 33277, SUNYaB A7A1-28, ATCC 53978, and 381) but did not hybridize with E. coli (JB45 and DH5α) or A. actinomycetemcomitans (ATCC 43718) (data not shown).

    Techniques: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Journal: Infection and Immunity

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    doi:

    Figure Lengend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Article Snippet: Under high-stringency conditions, this probe hybridized with DNA of all P. gingivalis strains (ATCC 33277, SUNYaB A7A1-28, ATCC 53978, and 381) but did not hybridize with E. coli (JB45 and DH5α) or A. actinomycetemcomitans (ATCC 43718) (data not shown).

    Techniques: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Journal: Infection and Immunity

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    doi:

    Figure Lengend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Article Snippet: Under high-stringency conditions, this probe hybridized with DNA of all P. gingivalis strains (ATCC 33277, SUNYaB A7A1-28, ATCC 53978, and 381) but did not hybridize with E. coli (JB45 and DH5α) or A. actinomycetemcomitans (ATCC 43718) (data not shown).

    Techniques: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Journal: Infection and Immunity

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    doi:

    Figure Lengend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Article Snippet: Under high-stringency conditions, this probe hybridized with DNA of all P. gingivalis strains (ATCC 33277, SUNYaB A7A1-28, ATCC 53978, and 381) but did not hybridize with E. coli (JB45 and DH5α) or A. actinomycetemcomitans (ATCC 43718) (data not shown).

    Techniques: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Journal: Infection and Immunity

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    doi:

    Figure Lengend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Article Snippet: Under high-stringency conditions, this probe hybridized with DNA of all P. gingivalis strains (ATCC 33277, SUNYaB A7A1-28, ATCC 53978, and 381) but did not hybridize with E. coli (JB45 and DH5α) or A. actinomycetemcomitans (ATCC 43718) (data not shown).

    Techniques: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Journal: Infection and Immunity

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    doi:

    Figure Lengend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Article Snippet: Under high-stringency conditions, this probe hybridized with DNA of all P. gingivalis strains (ATCC 33277, SUNYaB A7A1-28, ATCC 53978, and 381) but did not hybridize with E. coli (JB45 and DH5α) or A. actinomycetemcomitans (ATCC 43718) (data not shown).

    Techniques:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Journal: Infection and Immunity

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    doi:

    Figure Lengend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Article Snippet: Under high-stringency conditions, this probe hybridized with DNA of all P. gingivalis strains (ATCC 33277, SUNYaB A7A1-28, ATCC 53978, and 381) but did not hybridize with E. coli (JB45 and DH5α) or A. actinomycetemcomitans (ATCC 43718) (data not shown).

    Techniques: Cell Culture

    LPS downregulates cell membrane expression of TLR2 and TLR4. Gingival fibroblasts were treated with either E. coli or P. gingivalis ( P. ging ) LPS (50 ng/ml), collected at various times, fixed, stained without permeabilization with either anti-TLR2 (TLR2) or anti-TLR4 (TLR4) monoclonal antibody, and then stained with an alexa 488 nm-conjugated secondary antibody prior to flow cytometry analysis. Mean ± standard deviation; n = 4. (B) Preincubation of cultures with TLR2 and TLR4 blocking antibodies inhibited E. coli LPS stimulation of KGF-1 protein expression. Culture groups were treated with α-MEM with 1% FBS (control) or α-MEM with 1% FBS and 50 ng of LPS (LPS) per ml or first pretreated for 30 min with either 0.5 μg of anti-TLR2 (LPS+aT2) or TLR4 (LPS+aT4) blocking antibody per ml with α-MEM plus 1% FBS prior to the addition of LPS at 50 ng/ml. A sandwich ELISA was used to measure KGF-1 protein expression in 24-h conditioned medium. Mean ± standard deviation; n = 4.

    Journal: Infection and Immunity

    Article Title: Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4

    doi: 10.1128/IAI.70.12.6541-6548.2002

    Figure Lengend Snippet: LPS downregulates cell membrane expression of TLR2 and TLR4. Gingival fibroblasts were treated with either E. coli or P. gingivalis ( P. ging ) LPS (50 ng/ml), collected at various times, fixed, stained without permeabilization with either anti-TLR2 (TLR2) or anti-TLR4 (TLR4) monoclonal antibody, and then stained with an alexa 488 nm-conjugated secondary antibody prior to flow cytometry analysis. Mean ± standard deviation; n = 4. (B) Preincubation of cultures with TLR2 and TLR4 blocking antibodies inhibited E. coli LPS stimulation of KGF-1 protein expression. Culture groups were treated with α-MEM with 1% FBS (control) or α-MEM with 1% FBS and 50 ng of LPS (LPS) per ml or first pretreated for 30 min with either 0.5 μg of anti-TLR2 (LPS+aT2) or TLR4 (LPS+aT4) blocking antibody per ml with α-MEM plus 1% FBS prior to the addition of LPS at 50 ng/ml. A sandwich ELISA was used to measure KGF-1 protein expression in 24-h conditioned medium. Mean ± standard deviation; n = 4.

    Article Snippet: Cultures were treated with E. coli LPS (O55:B; Sigma catalog no. L2880) or P. gingivalis LPS (ATCC 33211) ( ) at 5 to 100 ng/ml or recombinant human sCD14 (R & D Systems Inc, Minneapolis, Minn.) at 50 ng/ml with α-MEM in the presence and absence of 1% FBS.

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Standard Deviation, Blocking Assay, Sandwich ELISA

    Illustration of a model for protective/destructive feedback through CD24-NLRP3 and P. gingivalis -NLRP3 interactions in healthy and diseased periodontal tissues. The non-keratinised lining epithelium (junctional epithelium) provides initial responses to bacterial products by signalling through receptors of innate immunity to activate NLRP3 inflammasome pathways (right panel). These comprise an intracellular network of regulatory and effector molecules leading to synthesis and activation of pro-inflammatory cytokines [IL-1β (green dots) IL-18 (red dots)]. However, activated IL-18 potently suppresses NLRP3 and release of IL-18. Destructive feedback accelerates periodontal tissue damage. Conversely, CD24 is characteristically strongly expressed in the lining epithelium of inflamed tissues and functions as an important negative regulator for response to danger signals, protecting tissues from excessive leukocyte activity mediated by microbial activity (left panel).

    Journal: Immunity, Inflammation and Disease

    Article Title: CD24 activates the NLRP3 inflammasome through c-Src kinase activity in a model of the lining epithelium of inflamed periodontal tissues

    doi: 10.1002/iid3.40

    Figure Lengend Snippet: Illustration of a model for protective/destructive feedback through CD24-NLRP3 and P. gingivalis -NLRP3 interactions in healthy and diseased periodontal tissues. The non-keratinised lining epithelium (junctional epithelium) provides initial responses to bacterial products by signalling through receptors of innate immunity to activate NLRP3 inflammasome pathways (right panel). These comprise an intracellular network of regulatory and effector molecules leading to synthesis and activation of pro-inflammatory cytokines [IL-1β (green dots) IL-18 (red dots)]. However, activated IL-18 potently suppresses NLRP3 and release of IL-18. Destructive feedback accelerates periodontal tissue damage. Conversely, CD24 is characteristically strongly expressed in the lining epithelium of inflamed tissues and functions as an important negative regulator for response to danger signals, protecting tissues from excessive leukocyte activity mediated by microbial activity (left panel).

    Article Snippet: RNA isolation and quantitative real-time RT-PCR for inflammasomes Confluent H413 clone-1 cells were incubated with one of the following conditions for 3 h: 5 µg/ml of CD24 mouse monoclonal (ALB9) peptide antibody (IgG1, GeneTex Inc USA), which recognises a short non-glycosylated peptide sequence close to the site of GPI linkage to the protein core of the cluster-w4/CD24 antigen ; treated with an IgG1 negative control antibody (5 µg/ml, DAKO, Denmark); treated with CD24 peptide antibody (5 µg/ml) plus c-Src inhibitor saracatinib (AZD0530, 1 µM); treated with recombinant IL-18 at 5 ng/ml in media; and treated with P. gingivalis strain (ATCC 33277) at a multiplicity of infection (MOI) of 100 .

    Techniques: Activation Assay, Activity Assay

    Quantitative real-time reverse transcription (RT)-PCR for gene expression of inflammasome and tight junction components in H413 epithelial cells in response to CD24 peptide antibody (a), active recombinant IL-18 (b and c), P. gingivalis (d) (* P

    Journal: Immunity, Inflammation and Disease

    Article Title: CD24 activates the NLRP3 inflammasome through c-Src kinase activity in a model of the lining epithelium of inflamed periodontal tissues

    doi: 10.1002/iid3.40

    Figure Lengend Snippet: Quantitative real-time reverse transcription (RT)-PCR for gene expression of inflammasome and tight junction components in H413 epithelial cells in response to CD24 peptide antibody (a), active recombinant IL-18 (b and c), P. gingivalis (d) (* P

    Article Snippet: RNA isolation and quantitative real-time RT-PCR for inflammasomes Confluent H413 clone-1 cells were incubated with one of the following conditions for 3 h: 5 µg/ml of CD24 mouse monoclonal (ALB9) peptide antibody (IgG1, GeneTex Inc USA), which recognises a short non-glycosylated peptide sequence close to the site of GPI linkage to the protein core of the cluster-w4/CD24 antigen ; treated with an IgG1 negative control antibody (5 µg/ml, DAKO, Denmark); treated with CD24 peptide antibody (5 µg/ml) plus c-Src inhibitor saracatinib (AZD0530, 1 µM); treated with recombinant IL-18 at 5 ng/ml in media; and treated with P. gingivalis strain (ATCC 33277) at a multiplicity of infection (MOI) of 100 .

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Recombinant

    Gingipain deficiency reverses the suppression of F. nucleatum invasion by P. gingivalis . HOK-16B cells were infected with CFSE-labeled F. nucleatum and unlabeled wild-type P. gingivalis or gingipain mutants at a MOI of 500 for 4 h. (A) Cells were analyzed using flow cytometry after quenching the extracellular fluorescence from the bacteria attached to the cell surface with trypan blue. The percentage of cells containing F. nucleatum (left panel) and the MFI (right panel) are shown as the mean ± standard deviation. Representative data from three independent experiments are shown. (B) The cells were examined under a confocal laser scanning microscope after staining for F-actin (red) and nuclei (blue). CFSE-labeled F. nucleatum is displayed in green. Fn, F. nucleatum ; Pg, P. gingivalis ; WT, P. gingivalis ATCC 33277; 129, KDP129 ( kgp − ); 133, KDP133 ( rgpA − rgpB − ); 136, KDP136 ( kgp − rgpA − rgpB − ). * p

    Journal: Journal of Oral Microbiology

    Article Title: Porphyromonas gingivalis suppresses invasion of Fusobacterium nucleatum into gingival epithelial cells

    doi: 10.1080/20002297.2017.1320193

    Figure Lengend Snippet: Gingipain deficiency reverses the suppression of F. nucleatum invasion by P. gingivalis . HOK-16B cells were infected with CFSE-labeled F. nucleatum and unlabeled wild-type P. gingivalis or gingipain mutants at a MOI of 500 for 4 h. (A) Cells were analyzed using flow cytometry after quenching the extracellular fluorescence from the bacteria attached to the cell surface with trypan blue. The percentage of cells containing F. nucleatum (left panel) and the MFI (right panel) are shown as the mean ± standard deviation. Representative data from three independent experiments are shown. (B) The cells were examined under a confocal laser scanning microscope after staining for F-actin (red) and nuclei (blue). CFSE-labeled F. nucleatum is displayed in green. Fn, F. nucleatum ; Pg, P. gingivalis ; WT, P. gingivalis ATCC 33277; 129, KDP129 ( kgp − ); 133, KDP133 ( rgpA − rgpB − ); 136, KDP136 ( kgp − rgpA − rgpB − ). * p

    Article Snippet: Wild-type P. gingivalis (ATCC 33277) and the mutant strains were grown anaerobically at 37°C in enriched BHI broth supplemented with 5 μg/mL of hemin and 1 μg/mL of vitamin K, and the following antibiotics, as described previously [ ]: chloramphenicol (20 μg/mL), erythromycin (10 μg/mL), and tetracycline (0.7 μg/mL).

    Techniques: Infection, Labeling, Flow Cytometry, Cytometry, Fluorescence, Standard Deviation, Laser-Scanning Microscopy, Staining

    P . gingivalis inactivates the PI3K/AKT pathway in a gingipain-dependent manner. (A) HOK-16B cells were infected with wild-type P. gingivalis at a MOI of 500 for the indicated time. (B) and (C) HOK-16B cells were infected with wild-type P. gingivalis or gingipain mutants in the presence or absence of F. nucleatum at a MOI of 500 for 30 min (B) or 3 h (C). The levels of phosphorylated and total forms of PI3K and AKT were analyzed by immunoblot analysis. Fn, F. nucleatum ; Pg, P. gingivalis ; WT, P. gingivalis ATCC 33277; 129, KDP129 ( kgp − ); 133, KDP133 ( rgpA − rgpB − ); 136, KDP136 ( kgp − rgpA − rgpB − ).

    Journal: Journal of Oral Microbiology

    Article Title: Porphyromonas gingivalis suppresses invasion of Fusobacterium nucleatum into gingival epithelial cells

    doi: 10.1080/20002297.2017.1320193

    Figure Lengend Snippet: P . gingivalis inactivates the PI3K/AKT pathway in a gingipain-dependent manner. (A) HOK-16B cells were infected with wild-type P. gingivalis at a MOI of 500 for the indicated time. (B) and (C) HOK-16B cells were infected with wild-type P. gingivalis or gingipain mutants in the presence or absence of F. nucleatum at a MOI of 500 for 30 min (B) or 3 h (C). The levels of phosphorylated and total forms of PI3K and AKT were analyzed by immunoblot analysis. Fn, F. nucleatum ; Pg, P. gingivalis ; WT, P. gingivalis ATCC 33277; 129, KDP129 ( kgp − ); 133, KDP133 ( rgpA − rgpB − ); 136, KDP136 ( kgp − rgpA − rgpB − ).

    Article Snippet: Wild-type P. gingivalis (ATCC 33277) and the mutant strains were grown anaerobically at 37°C in enriched BHI broth supplemented with 5 μg/mL of hemin and 1 μg/mL of vitamin K, and the following antibiotics, as described previously [ ]: chloramphenicol (20 μg/mL), erythromycin (10 μg/mL), and tetracycline (0.7 μg/mL).

    Techniques: Infection

    Invasion of F. nucleatum is suppressed by PI3K inhibition. (A) HOK-16B cells were preincubated with wortmannin at the indicated concentration for 30 min and then infected with CFSE-labeled F. nucleatum and unlabeled wild-type P. gingivalis at a MOI of 500 for 4 h. The percentage of cells containing F. nucleatum (left panel) and the MFI (right panel) are shown as the mean ± standard deviation. * p

    Journal: Journal of Oral Microbiology

    Article Title: Porphyromonas gingivalis suppresses invasion of Fusobacterium nucleatum into gingival epithelial cells

    doi: 10.1080/20002297.2017.1320193

    Figure Lengend Snippet: Invasion of F. nucleatum is suppressed by PI3K inhibition. (A) HOK-16B cells were preincubated with wortmannin at the indicated concentration for 30 min and then infected with CFSE-labeled F. nucleatum and unlabeled wild-type P. gingivalis at a MOI of 500 for 4 h. The percentage of cells containing F. nucleatum (left panel) and the MFI (right panel) are shown as the mean ± standard deviation. * p

    Article Snippet: Wild-type P. gingivalis (ATCC 33277) and the mutant strains were grown anaerobically at 37°C in enriched BHI broth supplemented with 5 μg/mL of hemin and 1 μg/mL of vitamin K, and the following antibiotics, as described previously [ ]: chloramphenicol (20 μg/mL), erythromycin (10 μg/mL), and tetracycline (0.7 μg/mL).

    Techniques: Inhibition, Concentration Assay, Infection, Labeling, Standard Deviation

    More intracellular F. nucleatum remains viable after coinfection with P. gingivalis with Rgp mutation than in monoinfection. After HOK-16B cells were infected with F. nucleatum in the presence or absence of wild-type P. gingivalis or gingipain mutants at a MOI of 500 for 4 h, extracellular bacteria were killed by incubation with antibiotics for 1 h. After incubation in fresh media for 12 h, the cells were lysed, and the lysates were plated on brain heart infusion blood agar plates containing vancomycin. The data represent the mean ± standard deviation of four independent experiments. * p

    Journal: Journal of Oral Microbiology

    Article Title: Porphyromonas gingivalis suppresses invasion of Fusobacterium nucleatum into gingival epithelial cells

    doi: 10.1080/20002297.2017.1320193

    Figure Lengend Snippet: More intracellular F. nucleatum remains viable after coinfection with P. gingivalis with Rgp mutation than in monoinfection. After HOK-16B cells were infected with F. nucleatum in the presence or absence of wild-type P. gingivalis or gingipain mutants at a MOI of 500 for 4 h, extracellular bacteria were killed by incubation with antibiotics for 1 h. After incubation in fresh media for 12 h, the cells were lysed, and the lysates were plated on brain heart infusion blood agar plates containing vancomycin. The data represent the mean ± standard deviation of four independent experiments. * p

    Article Snippet: Wild-type P. gingivalis (ATCC 33277) and the mutant strains were grown anaerobically at 37°C in enriched BHI broth supplemented with 5 μg/mL of hemin and 1 μg/mL of vitamin K, and the following antibiotics, as described previously [ ]: chloramphenicol (20 μg/mL), erythromycin (10 μg/mL), and tetracycline (0.7 μg/mL).

    Techniques: Mutagenesis, Infection, Incubation, Standard Deviation

    Sensitivity of P. gingivalis mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Sensitivity of P. gingivalis mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).

    Article Snippet: For construction of P. gingivalis dps mutants, ATCC 33277 and KDP139 were transformed to tetracycline resistance with linearized pKD388 ( dps ::Tcr ) DNA to yield KDP141 ( dps ::Tcr ) and KDP142 ( ftn ::Emr dps ::Tcr ), respectively.

    Techniques: Cell Culture, Concentration Assay, Incubation

    Sensitivity of P. gingivalis cells to hydrogen peroxide, mitomycin C, and metronidazole. P. gingivalis ATCC 33277 (wild type), KDP139 ( ftn ), KDP141 ( dps ), and KDP142 ( ftn dps ) were anaerobically grown in enriched BHI medium for 48 h. The cells were spread on enriched TS plates, and a paper disk containing hydrogen peroxide (A), mitomycin C (B), or metronidazole (C) was placed at the centers of the plates, followed by incubation at 37°C anaerobically for 7 days. The diameters of the clear zones next to the disks were measured (in millimeters). The data shown are the means and SD of triplicate experiments.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Sensitivity of P. gingivalis cells to hydrogen peroxide, mitomycin C, and metronidazole. P. gingivalis ATCC 33277 (wild type), KDP139 ( ftn ), KDP141 ( dps ), and KDP142 ( ftn dps ) were anaerobically grown in enriched BHI medium for 48 h. The cells were spread on enriched TS plates, and a paper disk containing hydrogen peroxide (A), mitomycin C (B), or metronidazole (C) was placed at the centers of the plates, followed by incubation at 37°C anaerobically for 7 days. The diameters of the clear zones next to the disks were measured (in millimeters). The data shown are the means and SD of triplicate experiments.

    Article Snippet: For construction of P. gingivalis dps mutants, ATCC 33277 and KDP139 were transformed to tetracycline resistance with linearized pKD388 ( dps ::Tcr ) DNA to yield KDP141 ( dps ::Tcr ) and KDP142 ( ftn ::Emr dps ::Tcr ), respectively.

    Techniques: Incubation

    DNA-binding activity of P. gingivalis Dps. Recombinant P. gingivalis Dps purified from the E. coli overexpressing P. gingivalis dps or the recombinant P. gingivalis Dps treated with ferrous ammonium sulfate was incubated with linear DNA (1-kb DNA ladder) at 4°C for 1 h. The mixture was then subjected to agarose gel electrophoresis. DNA on the gel was stained with ethidium bromide. Lanes: 1, DNA (500 ng) alone; 2, recombinant Dps (10 μg) alone; 3, recombinant Dps (10 μg) and DNA (500 ng); 4, iron-loaded recombinant Dps (10 μg) and DNA (500 ng); 5, iron-loaded recombinant Dps (10 μg) alone.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: DNA-binding activity of P. gingivalis Dps. Recombinant P. gingivalis Dps purified from the E. coli overexpressing P. gingivalis dps or the recombinant P. gingivalis Dps treated with ferrous ammonium sulfate was incubated with linear DNA (1-kb DNA ladder) at 4°C for 1 h. The mixture was then subjected to agarose gel electrophoresis. DNA on the gel was stained with ethidium bromide. Lanes: 1, DNA (500 ng) alone; 2, recombinant Dps (10 μg) alone; 3, recombinant Dps (10 μg) and DNA (500 ng); 4, iron-loaded recombinant Dps (10 μg) and DNA (500 ng); 5, iron-loaded recombinant Dps (10 μg) alone.

    Article Snippet: For construction of P. gingivalis dps mutants, ATCC 33277 and KDP139 were transformed to tetracycline resistance with linearized pKD388 ( dps ::Tcr ) DNA to yield KDP141 ( dps ::Tcr ) and KDP142 ( ftn ::Emr dps ::Tcr ), respectively.

    Techniques: Binding Assay, Activity Assay, Recombinant, Purification, Incubation, Agarose Gel Electrophoresis, Staining

    Alignment of the amino acid sequence of P. gingivalis Dps with those of the Dps previously isolated and determined in other prokaryotes.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Alignment of the amino acid sequence of P. gingivalis Dps with those of the Dps previously isolated and determined in other prokaryotes.

    Article Snippet: For construction of P. gingivalis dps mutants, ATCC 33277 and KDP139 were transformed to tetracycline resistance with linearized pKD388 ( dps ::Tcr ) DNA to yield KDP141 ( dps ::Tcr ) and KDP142 ( ftn ::Emr dps ::Tcr ), respectively.

    Techniques: Sequencing, Isolation

    Time course of induction of β-galactosidase activity in the dps′-′lacZ fusion strains. P. gingivalis KDP146 ( dps + dps′-′lacZ ) (circles) and KDP148 ( oxyR dps + dps′-′lacZ ) (triangles) were grown anaerobically in enriched BHI medium at 37°C. At an A 600 ). The β-galactosidase activity of the wild-type parent strain ATCC 33277 was

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Time course of induction of β-galactosidase activity in the dps′-′lacZ fusion strains. P. gingivalis KDP146 ( dps + dps′-′lacZ ) (circles) and KDP148 ( oxyR dps + dps′-′lacZ ) (triangles) were grown anaerobically in enriched BHI medium at 37°C. At an A 600 ). The β-galactosidase activity of the wild-type parent strain ATCC 33277 was

    Article Snippet: For construction of P. gingivalis dps mutants, ATCC 33277 and KDP139 were transformed to tetracycline resistance with linearized pKD388 ( dps ::Tcr ) DNA to yield KDP141 ( dps ::Tcr ) and KDP142 ( ftn ::Emr dps ::Tcr ), respectively.

    Techniques: Activity Assay

    Proof of authenticity of the P. gingivalis dps mutant KDP141 and the ftn dps double mutant KDP142. (A and B) Southern blot analyses of the chromosomal DNA. The chromosomal DNAs of the wild-type ATCC 33277 (lane 1) and the ftn mutants KDP139 (lane 2), KDP141 (lane 3), and KDP142 (lane 4) were digested with Nco I. The resulting DNA fragments were subjected to agarose gel electrophresis, followed by blotting. Hybridization was performed by using the 0.6-kb Nde I- Bgl II fragment of pKD390 as a dps probe (A) and the 2.7-kb Bam HI- Bgl II fragment of pKD375 as a tetQ probe (B). (C) Immunoblot analysis. After purified P. gingivalis Dps (lane 1) and the cell extracts of ATCC 33277 (lane 2), KDP141 (lane 3), and KDP142 (lane 4) were electrophoresed through an SDS-polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane and immunoreacted with antiserum against P. gingivalis Dps.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Proof of authenticity of the P. gingivalis dps mutant KDP141 and the ftn dps double mutant KDP142. (A and B) Southern blot analyses of the chromosomal DNA. The chromosomal DNAs of the wild-type ATCC 33277 (lane 1) and the ftn mutants KDP139 (lane 2), KDP141 (lane 3), and KDP142 (lane 4) were digested with Nco I. The resulting DNA fragments were subjected to agarose gel electrophresis, followed by blotting. Hybridization was performed by using the 0.6-kb Nde I- Bgl II fragment of pKD390 as a dps probe (A) and the 2.7-kb Bam HI- Bgl II fragment of pKD375 as a tetQ probe (B). (C) Immunoblot analysis. After purified P. gingivalis Dps (lane 1) and the cell extracts of ATCC 33277 (lane 2), KDP141 (lane 3), and KDP142 (lane 4) were electrophoresed through an SDS-polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane and immunoreacted with antiserum against P. gingivalis Dps.

    Article Snippet: For construction of P. gingivalis dps mutants, ATCC 33277 and KDP139 were transformed to tetracycline resistance with linearized pKD388 ( dps ::Tcr ) DNA to yield KDP141 ( dps ::Tcr ) and KDP142 ( ftn ::Emr dps ::Tcr ), respectively.

    Techniques: Mutagenesis, Southern Blot, Agarose Gel Electrophoresis, Hybridization, Purification

    Survival of P. gingivalis cells in HUVEC. Monolayers of HUVEC were infected by P. gingivalis ATCC 33277 (wild type) and KDP141 ( dps ). Experiments were done at least five times, and the data are presented as the mean and the SD of the CFU per HUVEC.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Survival of P. gingivalis cells in HUVEC. Monolayers of HUVEC were infected by P. gingivalis ATCC 33277 (wild type) and KDP141 ( dps ). Experiments were done at least five times, and the data are presented as the mean and the SD of the CFU per HUVEC.

    Article Snippet: For construction of P. gingivalis dps mutants, ATCC 33277 and KDP139 were transformed to tetracycline resistance with linearized pKD388 ( dps ::Tcr ) DNA to yield KDP141 ( dps ::Tcr ) and KDP142 ( ftn ::Emr dps ::Tcr ), respectively.

    Techniques: Infection

    Correlation between duplicate samples in HCN offline headspace measurements of P. gingivalis strains. One data point (▪) represents the HCN concentrations obtained from duplicate No. 1 (horizontal axis) and No. 2 (vertical axis). Each strain was measured at 24, 48 and 72 hours, hence, there were three data points for each strain, and a total of 12 points for all four strains. A strong correlation ( r s = 0.96 and p

    Journal: Scientific Reports

    Article Title: Detection of hydrogen cyanide from oral anaerobes by cavity ring down spectroscopy

    doi: 10.1038/srep22577

    Figure Lengend Snippet: Correlation between duplicate samples in HCN offline headspace measurements of P. gingivalis strains. One data point (▪) represents the HCN concentrations obtained from duplicate No. 1 (horizontal axis) and No. 2 (vertical axis). Each strain was measured at 24, 48 and 72 hours, hence, there were three data points for each strain, and a total of 12 points for all four strains. A strong correlation ( r s = 0.96 and p

    Article Snippet: Further investigations were conducted on the species of P. gingivalis and we successfully detected HCN production (0.9–10.9 ppb) in the headspace of three P. gingivalis reference strains (ATCC 33277, W50 and OMG 434) and one clinical isolate.

    Techniques:

    Detection of HCN and CO 2 from P. gingivalis strains and blank Brucella blood agar. The P. gingivalis strains included three reference strains ( a ) ATCC 33277, ( b ) W50, ( c ) OMG 434 and one clinical isolate ( d ) 4753E. Blank Brucella blood agar ( e ) served as background control. There were duplicate samples for each strain. The detection was conducted at 24, 48 and 72 hours.

    Journal: Scientific Reports

    Article Title: Detection of hydrogen cyanide from oral anaerobes by cavity ring down spectroscopy

    doi: 10.1038/srep22577

    Figure Lengend Snippet: Detection of HCN and CO 2 from P. gingivalis strains and blank Brucella blood agar. The P. gingivalis strains included three reference strains ( a ) ATCC 33277, ( b ) W50, ( c ) OMG 434 and one clinical isolate ( d ) 4753E. Blank Brucella blood agar ( e ) served as background control. There were duplicate samples for each strain. The detection was conducted at 24, 48 and 72 hours.

    Article Snippet: Further investigations were conducted on the species of P. gingivalis and we successfully detected HCN production (0.9–10.9 ppb) in the headspace of three P. gingivalis reference strains (ATCC 33277, W50 and OMG 434) and one clinical isolate.

    Techniques:

    Dynamic profiles of HCN production by different P. gingivalis strains. The HCN concentrations from three reference strains (ATCC 33277, W50 and OMG 434) and one clinical isolate (4753E) of P. gingivalis were measured. The HCN concentrations from each strain were measured every 20 minutes. For clarity, data points are shown only every two hours.

    Journal: Scientific Reports

    Article Title: Detection of hydrogen cyanide from oral anaerobes by cavity ring down spectroscopy

    doi: 10.1038/srep22577

    Figure Lengend Snippet: Dynamic profiles of HCN production by different P. gingivalis strains. The HCN concentrations from three reference strains (ATCC 33277, W50 and OMG 434) and one clinical isolate (4753E) of P. gingivalis were measured. The HCN concentrations from each strain were measured every 20 minutes. For clarity, data points are shown only every two hours.

    Article Snippet: Further investigations were conducted on the species of P. gingivalis and we successfully detected HCN production (0.9–10.9 ppb) in the headspace of three P. gingivalis reference strains (ATCC 33277, W50 and OMG 434) and one clinical isolate.

    Techniques:

    Correlation between HCN and CO 2 concentrations of P. gingivalis ATCC 33277 and W50. Data points (▪) represent the concentration of HCN (horizontal axis) and CO 2 (vertical axis) determined from one plate at a certain time point. Since each strain was measured in duplicate and at 24, 48 and 72 hours, each strain has six data points. In total, there are 12 data points for two reference strains, P. gingivalis ATCC 33277 and W50. A strong correlation ( r s = 0.89 and p

    Journal: Scientific Reports

    Article Title: Detection of hydrogen cyanide from oral anaerobes by cavity ring down spectroscopy

    doi: 10.1038/srep22577

    Figure Lengend Snippet: Correlation between HCN and CO 2 concentrations of P. gingivalis ATCC 33277 and W50. Data points (▪) represent the concentration of HCN (horizontal axis) and CO 2 (vertical axis) determined from one plate at a certain time point. Since each strain was measured in duplicate and at 24, 48 and 72 hours, each strain has six data points. In total, there are 12 data points for two reference strains, P. gingivalis ATCC 33277 and W50. A strong correlation ( r s = 0.89 and p

    Article Snippet: Further investigations were conducted on the species of P. gingivalis and we successfully detected HCN production (0.9–10.9 ppb) in the headspace of three P. gingivalis reference strains (ATCC 33277, W50 and OMG 434) and one clinical isolate.

    Techniques: Concentration Assay

    TBARS analysis of LDL incubated with CuSO 4 and HDL in the presence and absence of Porphyromonas gingivalis . LDL: low-density lipoprotein, HDL: high-density lipoprotein, Control: sample without LDL, HDL, and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis , TBARS: thiobarbituric acid-reactive substances. a) Statistically significant ( P

    Journal: Journal of Periodontal & Implant Science

    Article Title: Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein

    doi: 10.5051/jpis.2018.48.1.60

    Figure Lengend Snippet: TBARS analysis of LDL incubated with CuSO 4 and HDL in the presence and absence of Porphyromonas gingivalis . LDL: low-density lipoprotein, HDL: high-density lipoprotein, Control: sample without LDL, HDL, and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis , TBARS: thiobarbituric acid-reactive substances. a) Statistically significant ( P

    Article Snippet: Bacteria culture and HDL preparation P. gingivalis (ATCC 33277, American Type Culture Collection, Manassas, VA, USA) was cultured anaerobically in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) supplemented with hemin and menadione.

    Techniques: Incubation

    Quantification of Porphyromonas gingivalis -induced HDL oxidation using a TBARS assay. HDL: high-density lipoprotein, TBARS: thiobarbituric acid-reactive substances, Control: sample without HDL and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis . a) Statistically significant ( P

    Journal: Journal of Periodontal & Implant Science

    Article Title: Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein

    doi: 10.5051/jpis.2018.48.1.60

    Figure Lengend Snippet: Quantification of Porphyromonas gingivalis -induced HDL oxidation using a TBARS assay. HDL: high-density lipoprotein, TBARS: thiobarbituric acid-reactive substances, Control: sample without HDL and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis . a) Statistically significant ( P

    Article Snippet: Bacteria culture and HDL preparation P. gingivalis (ATCC 33277, American Type Culture Collection, Manassas, VA, USA) was cultured anaerobically in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) supplemented with hemin and menadione.

    Techniques: TBARS Assay

    Proinflammatory activity by Porphyromonas gingivalis -induced oxidized HDL. (A) Monocytes incubated with Porphyromonas gingivalis in the presence of HDL produced significantly higher levels of TNF-α than monocytes treated with CuSO 4 or with HDL alone. (B) Monocytes incubated with Porphyromonas gingivalis and HDL showed higher MMP-9 activity than cells incubated with HDL alone. HDL: high-density lipoprotein, TNF-α: tumor necrosis factor alpha, MMP: matrix metalloproteinase, P.g. : Porphyromonas gingivalis , Control: sample without Porphyromonas gingivalis . a) Statistically significant ( P

    Journal: Journal of Periodontal & Implant Science

    Article Title: Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein

    doi: 10.5051/jpis.2018.48.1.60

    Figure Lengend Snippet: Proinflammatory activity by Porphyromonas gingivalis -induced oxidized HDL. (A) Monocytes incubated with Porphyromonas gingivalis in the presence of HDL produced significantly higher levels of TNF-α than monocytes treated with CuSO 4 or with HDL alone. (B) Monocytes incubated with Porphyromonas gingivalis and HDL showed higher MMP-9 activity than cells incubated with HDL alone. HDL: high-density lipoprotein, TNF-α: tumor necrosis factor alpha, MMP: matrix metalloproteinase, P.g. : Porphyromonas gingivalis , Control: sample without Porphyromonas gingivalis . a) Statistically significant ( P

    Article Snippet: Bacteria culture and HDL preparation P. gingivalis (ATCC 33277, American Type Culture Collection, Manassas, VA, USA) was cultured anaerobically in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) supplemented with hemin and menadione.

    Techniques: Activity Assay, Incubation, Produced

    Macrophage foam cell formation assessed by Oil Red O staining. The uptake of oxidized HDL by macrophages treated with (A) HDL only, (B) HDL oxidized with Porphyromonas gingivalis , and (C) HDL oxidized with CuSO 4 was determined by light microscopy (scale bar=200 μm). HDL: high-density lipoprotein.

    Journal: Journal of Periodontal & Implant Science

    Article Title: Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein

    doi: 10.5051/jpis.2018.48.1.60

    Figure Lengend Snippet: Macrophage foam cell formation assessed by Oil Red O staining. The uptake of oxidized HDL by macrophages treated with (A) HDL only, (B) HDL oxidized with Porphyromonas gingivalis , and (C) HDL oxidized with CuSO 4 was determined by light microscopy (scale bar=200 μm). HDL: high-density lipoprotein.

    Article Snippet: Bacteria culture and HDL preparation P. gingivalis (ATCC 33277, American Type Culture Collection, Manassas, VA, USA) was cultured anaerobically in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) supplemented with hemin and menadione.

    Techniques: Staining, Light Microscopy

    Dilution curves of mouse plasma IgM binding to antigens. A, B) C57BL/6 mice were immunized with heat-killed P. gingivalis ATCC33277 (Pg) and controls (Co) received saline. The plasma was diluted 1∶100–1∶6400 and IgM binding to MDA-LDL, CuOx-LDL, native LDL, PC-BSA (A), P. gingivalis and recombinant gingipain domains Rgp44, Rgp15–27 and RgpCAT (B) were determined before (pre) and after (post) immunization with chemiluminescence immunoassay. Mean ± SD for two samples is shown. C, D) LDLR −/− mice were immunized with killed P. gingivalis (3 strains mixed) (Pg) and controls (Co) received PBS. Pooled plasma from two mice was used for each dilution curve, and mean ± SD for two dilution curves is shown. IgM binding to MDA-LDL, CuOx-LDL, native LDL, PC-BSA (C), P. gingivalis and recombinant gingipain domain Rgp44, Rgp15–27 and RgpCAT (D) were determined before (pre) and after the second booster immunization (imm) as described.

    Journal: PLoS ONE

    Article Title: Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0034910

    Figure Lengend Snippet: Dilution curves of mouse plasma IgM binding to antigens. A, B) C57BL/6 mice were immunized with heat-killed P. gingivalis ATCC33277 (Pg) and controls (Co) received saline. The plasma was diluted 1∶100–1∶6400 and IgM binding to MDA-LDL, CuOx-LDL, native LDL, PC-BSA (A), P. gingivalis and recombinant gingipain domains Rgp44, Rgp15–27 and RgpCAT (B) were determined before (pre) and after (post) immunization with chemiluminescence immunoassay. Mean ± SD for two samples is shown. C, D) LDLR −/− mice were immunized with killed P. gingivalis (3 strains mixed) (Pg) and controls (Co) received PBS. Pooled plasma from two mice was used for each dilution curve, and mean ± SD for two dilution curves is shown. IgM binding to MDA-LDL, CuOx-LDL, native LDL, PC-BSA (C), P. gingivalis and recombinant gingipain domain Rgp44, Rgp15–27 and RgpCAT (D) were determined before (pre) and after the second booster immunization (imm) as described.

    Article Snippet: Female C57BL/6 mice on chow diet (n = 8) were immunized without adjuvant with one strain of heat-killed P. gingivalis (ATCC 33277, 2×108 CFU) in sterile saline injected subcutaneously.

    Techniques: Binding Assay, Mouse Assay, Multiple Displacement Amplification, Recombinant, Chemiluminescence Immunoassay

    Mouse plasma IgM and IgG binding to MDA-LDL after immunization with P. gingivalis . C57BL/6 mice were immunized with heat-killed P. gingivalis ATCC33277 (Pg; n = 8) and controls received saline (Co; n = 8). Plasma IgM (A) and IgG (B) to MDA-LDL before (pre) and after immunization (post) were determined with chemiluminescence immunoassay. Each C57BL/6 plasma sample (1∶500) was measured in duplicate and an average for each individual was calculated. LDLR −/− mice were immunized with killed P. gingivalis (3 strains mixed) (Pg; n = 7) and controls received PBS (Co; n = 8). Plasma IgM (C) and IgG (D) to MDA-LDL after the second booster immunization (imm) and after the HFD (end) were determined. Each LDLR −/− plasma sample (1∶1000) was measured in duplicate and an average for each individual in two repeated assays was calculated. Additionally, mouse plasma IgM binding to CuOx-LDL (E, G) and PC-BSA (F, H) was determined. For C57BL/6 mice (E, F) this was done similarly as described for panel A. Plasma samples of LDLR −/− mice (G, H) were pooled between three or four mice (1∶1000) for a single assay, in which the mean ± SD within a group is shown.

    Journal: PLoS ONE

    Article Title: Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0034910

    Figure Lengend Snippet: Mouse plasma IgM and IgG binding to MDA-LDL after immunization with P. gingivalis . C57BL/6 mice were immunized with heat-killed P. gingivalis ATCC33277 (Pg; n = 8) and controls received saline (Co; n = 8). Plasma IgM (A) and IgG (B) to MDA-LDL before (pre) and after immunization (post) were determined with chemiluminescence immunoassay. Each C57BL/6 plasma sample (1∶500) was measured in duplicate and an average for each individual was calculated. LDLR −/− mice were immunized with killed P. gingivalis (3 strains mixed) (Pg; n = 7) and controls received PBS (Co; n = 8). Plasma IgM (C) and IgG (D) to MDA-LDL after the second booster immunization (imm) and after the HFD (end) were determined. Each LDLR −/− plasma sample (1∶1000) was measured in duplicate and an average for each individual in two repeated assays was calculated. Additionally, mouse plasma IgM binding to CuOx-LDL (E, G) and PC-BSA (F, H) was determined. For C57BL/6 mice (E, F) this was done similarly as described for panel A. Plasma samples of LDLR −/− mice (G, H) were pooled between three or four mice (1∶1000) for a single assay, in which the mean ± SD within a group is shown.

    Article Snippet: Female C57BL/6 mice on chow diet (n = 8) were immunized without adjuvant with one strain of heat-killed P. gingivalis (ATCC 33277, 2×108 CFU) in sterile saline injected subcutaneously.

    Techniques: Binding Assay, Multiple Displacement Amplification, Mouse Assay, Chemiluminescence Immunoassay

    Quantification of atherosclerosis in LDLR −/− mice immunized with P. gingivalis . LDLR −/− mice (n = 7) were immunized without adjuvant with killed P. gingivalis (3 strains mixed) (Pg) followed by high fat diet (HFD). Controls (PBS, n = 8) received PBS. A) The extent of atherosclerotic plaque development was determined after HFD by en face analysis of the Sudan IV -stained aortas. B) Lesions at the aortic origin were measured on histological sections as percentage of plaque area in the aorta cross-sectional area. Representative pictures of aortas and cross-sections are shown for each group. * P

    Journal: PLoS ONE

    Article Title: Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0034910

    Figure Lengend Snippet: Quantification of atherosclerosis in LDLR −/− mice immunized with P. gingivalis . LDLR −/− mice (n = 7) were immunized without adjuvant with killed P. gingivalis (3 strains mixed) (Pg) followed by high fat diet (HFD). Controls (PBS, n = 8) received PBS. A) The extent of atherosclerotic plaque development was determined after HFD by en face analysis of the Sudan IV -stained aortas. B) Lesions at the aortic origin were measured on histological sections as percentage of plaque area in the aorta cross-sectional area. Representative pictures of aortas and cross-sections are shown for each group. * P

    Article Snippet: Female C57BL/6 mice on chow diet (n = 8) were immunized without adjuvant with one strain of heat-killed P. gingivalis (ATCC 33277, 2×108 CFU) in sterile saline injected subcutaneously.

    Techniques: Mouse Assay, Staining

    Distinction of PPAD sorting types I and II P. gingivalis isolates were cultured for four days in BHI medium. Subsequently, bacterial cells were separated from the growth medium, and growth medium fractions, containing OMVs, were used for immunoblotting with PPAD-specific antibodies. (A) P. gingivalis reference strain W83 and the isogenic PPAD deletion mutant. (B) P. gingivalis clinical isolates. Names of sorting type II isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated.

    Journal: Virulence

    Article Title: There's no place like OM: Vesicular sorting and secretion of the peptidylarginine deiminase of Porphyromonas gingivalis

    doi: 10.1080/21505594.2017.1421827

    Figure Lengend Snippet: Distinction of PPAD sorting types I and II P. gingivalis isolates were cultured for four days in BHI medium. Subsequently, bacterial cells were separated from the growth medium, and growth medium fractions, containing OMVs, were used for immunoblotting with PPAD-specific antibodies. (A) P. gingivalis reference strain W83 and the isogenic PPAD deletion mutant. (B) P. gingivalis clinical isolates. Names of sorting type II isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated.

    Article Snippet: Additionally, the study isolates included one P. gingivalis isolate from a healthy carrier, and two P. gingivalis type strains (W83 and ATCC 33277), with the respective engineered PPAD deletion mutants [ ].

    Techniques: Cell Culture, Mutagenesis, Marker

    OMV formation by sorting type I and II isolates of P. gingivalis Electron micrographs of vesiculating cells of (A) the P. gingivalis type strain W83, and (B) the sorting type II isolate MDS33. Electron micrographs of OMVs collected from (C) strain W83, (D) the sorting type I isolate 505700, and the sorting type II isolates (E) 505759 and (F) 512915.

    Journal: Virulence

    Article Title: There's no place like OM: Vesicular sorting and secretion of the peptidylarginine deiminase of Porphyromonas gingivalis

    doi: 10.1080/21505594.2017.1421827

    Figure Lengend Snippet: OMV formation by sorting type I and II isolates of P. gingivalis Electron micrographs of vesiculating cells of (A) the P. gingivalis type strain W83, and (B) the sorting type II isolate MDS33. Electron micrographs of OMVs collected from (C) strain W83, (D) the sorting type I isolate 505700, and the sorting type II isolates (E) 505759 and (F) 512915.

    Article Snippet: Additionally, the study isolates included one P. gingivalis isolate from a healthy carrier, and two P. gingivalis type strains (W83 and ATCC 33277), with the respective engineered PPAD deletion mutants [ ].

    Techniques:

    Position of amino acid substitutions in PPAD and their impact on the electrostatic potential of the protein (A) 3D-structural ribbon representation of the PPAD protein from P. gingivalis reference strain W83, showing in yellow surface-exposed amino acid residues that have been substituted in PPAD proteins from other P. gingivalis sorting type II isolates. Catalytic residues of PPAD are shown in green. (B and C) Electrostatic potential maps showing, respectively, the PPAD proteins of strain W83 and the sorting type II isolate MDS33 from the same perspective. The two maps display the difference in electrostatic potential (red represents -5 KT/e and blue +5 KT/e ), and the respective Gln or Lys residues at position 373 are indicated. (D) 3D-structural ribbon representation of the PPAD protein from P. gingivalis strain W83, showing surface-exposed amino acid residues that have been substituted in other PPAD proteins (marked in yellow) of sorting type II isolates from the perspective of the catalytic site. The catalytic residues are marked in green. (E) Electrostatic potential map of the PPAD protein of strain W83, displaying all the residues subject to substitutions in sorting type II isolates. (F) Electrostatic potential map of the PPAD protein from the sorting type II isolate MDS33.

    Journal: Virulence

    Article Title: There's no place like OM: Vesicular sorting and secretion of the peptidylarginine deiminase of Porphyromonas gingivalis

    doi: 10.1080/21505594.2017.1421827

    Figure Lengend Snippet: Position of amino acid substitutions in PPAD and their impact on the electrostatic potential of the protein (A) 3D-structural ribbon representation of the PPAD protein from P. gingivalis reference strain W83, showing in yellow surface-exposed amino acid residues that have been substituted in PPAD proteins from other P. gingivalis sorting type II isolates. Catalytic residues of PPAD are shown in green. (B and C) Electrostatic potential maps showing, respectively, the PPAD proteins of strain W83 and the sorting type II isolate MDS33 from the same perspective. The two maps display the difference in electrostatic potential (red represents -5 KT/e and blue +5 KT/e ), and the respective Gln or Lys residues at position 373 are indicated. (D) 3D-structural ribbon representation of the PPAD protein from P. gingivalis strain W83, showing surface-exposed amino acid residues that have been substituted in other PPAD proteins (marked in yellow) of sorting type II isolates from the perspective of the catalytic site. The catalytic residues are marked in green. (E) Electrostatic potential map of the PPAD protein of strain W83, displaying all the residues subject to substitutions in sorting type II isolates. (F) Electrostatic potential map of the PPAD protein from the sorting type II isolate MDS33.

    Article Snippet: Additionally, the study isolates included one P. gingivalis isolate from a healthy carrier, and two P. gingivalis type strains (W83 and ATCC 33277), with the respective engineered PPAD deletion mutants [ ].

    Techniques:

    Evaluation of antimicrobial activity of the MNA-TNTs, GL13K-TNTs, and TNTs against ( A ) Porphyromonas gingivalis ATCC 33277 and ( B ) Fusobacterium nucleatum ATCC 25586, assessed using disk-diffusion assay. Abbreviations: MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO 2 nanotubes; ATCC, American Type Culture Collection.

    Journal: International Journal of Nanomedicine

    Article Title: Antibacterial activity and cytocompatibility of an implant coating consisting of TiO2 nanotubes combined with a GL13K antimicrobial peptide

    doi: 10.2147/IJN.S128775

    Figure Lengend Snippet: Evaluation of antimicrobial activity of the MNA-TNTs, GL13K-TNTs, and TNTs against ( A ) Porphyromonas gingivalis ATCC 33277 and ( B ) Fusobacterium nucleatum ATCC 25586, assessed using disk-diffusion assay. Abbreviations: MNA, metronidazole; MNA-TNTs, MNA-immobilized TNTs; TNTs, TiO 2 nanotubes; ATCC, American Type Culture Collection.

    Article Snippet: Antimicrobial activity assessment The antimicrobial activities of the specimens against two Gram-negative anaerobic bacterial strains, F. nucleatum (American Type Culture Collection [ATCC] 25586) and P. gingivalis (ATCC 33277), were assessed using a disk-diffusion assay (Kirby–Bauer).

    Techniques: Activity Assay, Diffusion-based Assay

    Fluorescence -microscopic analysis of co-localization between vacuolar- and cytosolic- P. gingivalis with NDP52 ubiquitin-binding-adaptor protein in primary GECs. (A) Primary GECs were infected with PgFbFP (green fluorescence) and after differential digitonin permeabilization stained with an anti- P. gingivalis antibody (Alexa 594, red fluorescence), followed by staining against NDP52 (Alexa 350, blue fluorescence). Cytoplasmic bacteria were detected as FbFP-and Alexa Fluor 594 positive (yellow) previously, whereas vacuolar bacteria were only FbFP-positive (green). 40x micrographs; Bar 10 µm. (B) Majority of cytosolic P. gingivalis was determined to show a significant association with NDP52 (red and blue = purple). Mean +/− SD, n > 3.

    Journal: Virulence

    Article Title: Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells

    doi: 10.1080/21505594.2018.1454171

    Figure Lengend Snippet: Fluorescence -microscopic analysis of co-localization between vacuolar- and cytosolic- P. gingivalis with NDP52 ubiquitin-binding-adaptor protein in primary GECs. (A) Primary GECs were infected with PgFbFP (green fluorescence) and after differential digitonin permeabilization stained with an anti- P. gingivalis antibody (Alexa 594, red fluorescence), followed by staining against NDP52 (Alexa 350, blue fluorescence). Cytoplasmic bacteria were detected as FbFP-and Alexa Fluor 594 positive (yellow) previously, whereas vacuolar bacteria were only FbFP-positive (green). 40x micrographs; Bar 10 µm. (B) Majority of cytosolic P. gingivalis was determined to show a significant association with NDP52 (red and blue = purple). Mean +/− SD, n > 3.

    Article Snippet: Cells were then incubated with blocking buffer (PBS, 0.1% Triton-X, 2% BSA) for 20 minutes, followed by incubation of primary antibodies of rabbit anti- P. gingivalis ATCC 33277 1:1000, mouse anti-LC3 1:1000 (Cell Signaling) or goat anti-Bip (1:1000) (Santa Cruz).

    Techniques: Fluorescence, Binding Assay, Infection, Staining

    P. gingivalis vacuolar localization significantly increases over time of infection in primary GECs. Immunofluorescence intensity based quantification of cytoplasmic and vacuolar P. gingivalis at 3, 6, 12, and 24 hours post infection. (A) Primary GECs were infected with PgFbFP, green fluorescing P. gingivalis at MOI 100. Infected PgFbFP were labeled using anti- P. gingivalis antibody followed by Alexa 594 (red-fluorescence) secondary antibody after selective digitonin permeabilization (which will only permeabilize cellular plasma membrane). Cytoplasmic bacteria were detected as FbFP-and Alexa 594 positive (yellow), whereas vacuolar bacteria were solely FbFP-positive (green). 40x micrographs; Bar 10 µm. (B) The percentage of intracellular bacteria are represented as mean +/− SD; n > 3. p

    Journal: Virulence

    Article Title: Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells

    doi: 10.1080/21505594.2018.1454171

    Figure Lengend Snippet: P. gingivalis vacuolar localization significantly increases over time of infection in primary GECs. Immunofluorescence intensity based quantification of cytoplasmic and vacuolar P. gingivalis at 3, 6, 12, and 24 hours post infection. (A) Primary GECs were infected with PgFbFP, green fluorescing P. gingivalis at MOI 100. Infected PgFbFP were labeled using anti- P. gingivalis antibody followed by Alexa 594 (red-fluorescence) secondary antibody after selective digitonin permeabilization (which will only permeabilize cellular plasma membrane). Cytoplasmic bacteria were detected as FbFP-and Alexa 594 positive (yellow), whereas vacuolar bacteria were solely FbFP-positive (green). 40x micrographs; Bar 10 µm. (B) The percentage of intracellular bacteria are represented as mean +/− SD; n > 3. p

    Article Snippet: Cells were then incubated with blocking buffer (PBS, 0.1% Triton-X, 2% BSA) for 20 minutes, followed by incubation of primary antibodies of rabbit anti- P. gingivalis ATCC 33277 1:1000, mouse anti-LC3 1:1000 (Cell Signaling) or goat anti-Bip (1:1000) (Santa Cruz).

    Techniques: Infection, Immunofluorescence, Labeling, Fluorescence

    Fluorescence microscopic analysis of co-localization between vacuolar- and cytosolic- P. gingivalis with p62 ubiquitin-binding-adaptor protein in primary GECs. (A) Primary GECs were infected with PgFbFP (green fluorescence) and after differential digitonin permeabilization stained with an anti- P. gingivalis antibody (Alexa 594, red fluorescence), followed by staining against p62 antibody (Alexa 350, blue fluorescence). Cytoplasmic bacteria were detected as FbFP-and Alexa Fluor 594 positive (yellow) previously, whereas vacuolar bacteria were only FbFP-positive (green). 40x micrographs; Bar 10 µm. (B) Majority of cytosolic P. gingivalis was determined to show a significant association with p62 (red and blue = purple). Mean +/− SD, n > 3.

    Journal: Virulence

    Article Title: Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells

    doi: 10.1080/21505594.2018.1454171

    Figure Lengend Snippet: Fluorescence microscopic analysis of co-localization between vacuolar- and cytosolic- P. gingivalis with p62 ubiquitin-binding-adaptor protein in primary GECs. (A) Primary GECs were infected with PgFbFP (green fluorescence) and after differential digitonin permeabilization stained with an anti- P. gingivalis antibody (Alexa 594, red fluorescence), followed by staining against p62 antibody (Alexa 350, blue fluorescence). Cytoplasmic bacteria were detected as FbFP-and Alexa Fluor 594 positive (yellow) previously, whereas vacuolar bacteria were only FbFP-positive (green). 40x micrographs; Bar 10 µm. (B) Majority of cytosolic P. gingivalis was determined to show a significant association with p62 (red and blue = purple). Mean +/− SD, n > 3.

    Article Snippet: Cells were then incubated with blocking buffer (PBS, 0.1% Triton-X, 2% BSA) for 20 minutes, followed by incubation of primary antibodies of rabbit anti- P. gingivalis ATCC 33277 1:1000, mouse anti-LC3 1:1000 (Cell Signaling) or goat anti-Bip (1:1000) (Santa Cruz).

    Techniques: Fluorescence, Binding Assay, Infection, Staining

    Proliferation and differentiation of CD4 + T cells mediated by gingipains from P. gingivalis . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence/absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) or the isogenic gingipain-null mutant ΔKΔRAB for 6 h at MOI (multiplicity of infection) 1:50. Naive CD4 + T cells stained with CFSE were added and co-stimulated for 5 days. Proliferation of lymphocytes was measured at day 1, 3, and 5. (A) Completed results of proliferation on day 1, 3, and 5 as a percentage of CFSE low cells. Data are presented as a mean ± standard deviation of assays performed in triplicate using four independent donors, and were analyzed with a two way ANOVA with the Bonferoni's posttest correction (# P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

    doi: 10.3389/fcimb.2017.00140

    Figure Lengend Snippet: Proliferation and differentiation of CD4 + T cells mediated by gingipains from P. gingivalis . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence/absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) or the isogenic gingipain-null mutant ΔKΔRAB for 6 h at MOI (multiplicity of infection) 1:50. Naive CD4 + T cells stained with CFSE were added and co-stimulated for 5 days. Proliferation of lymphocytes was measured at day 1, 3, and 5. (A) Completed results of proliferation on day 1, 3, and 5 as a percentage of CFSE low cells. Data are presented as a mean ± standard deviation of assays performed in triplicate using four independent donors, and were analyzed with a two way ANOVA with the Bonferoni's posttest correction (# P

    Article Snippet: Stimulation of moDCS with virulence factors from P. gingivalis Dendritic cells at a concentration of 0.6 × 106 /ml were left untreated (control) or stimulated with Ultra Pure LPS from P. gingivalis (InvivoGen) [2.5 μg/ml], FimA purified from ATCC 33277 [10 μg/ml] or both together.

    Techniques: Derivative Assay, Concentration Assay, Mutagenesis, Infection, Staining, Standard Deviation

    Activation of Th17 signaling pathway in CD4+ naïve lymphocyte depends on gingipain activity . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM). After 6 h, CD4+ naïve lymphocytes were added and co-stimulated for 3 consecutive days. At day 1, 2, and 3 after co-incubation, cells were collected and lysed with TRIzol, RNA was isolated and reverse transcriptase PCR was performed. Relative expression of cytokine genes IL-6R, IL-1R, TGF β R, RORc, ROR α , STAT3, RUNX1, IRF4, BATF to the reference house-keeping gene EF2 was measured by Real-Time PCR. Data represent fold increase in expression compared to control levels, which were arbitrarily set at 1 and were analyzed with a Student's t -test ( # P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

    doi: 10.3389/fcimb.2017.00140

    Figure Lengend Snippet: Activation of Th17 signaling pathway in CD4+ naïve lymphocyte depends on gingipain activity . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM). After 6 h, CD4+ naïve lymphocytes were added and co-stimulated for 3 consecutive days. At day 1, 2, and 3 after co-incubation, cells were collected and lysed with TRIzol, RNA was isolated and reverse transcriptase PCR was performed. Relative expression of cytokine genes IL-6R, IL-1R, TGF β R, RORc, ROR α , STAT3, RUNX1, IRF4, BATF to the reference house-keeping gene EF2 was measured by Real-Time PCR. Data represent fold increase in expression compared to control levels, which were arbitrarily set at 1 and were analyzed with a Student's t -test ( # P

    Article Snippet: Stimulation of moDCS with virulence factors from P. gingivalis Dendritic cells at a concentration of 0.6 × 106 /ml were left untreated (control) or stimulated with Ultra Pure LPS from P. gingivalis (InvivoGen) [2.5 μg/ml], FimA purified from ATCC 33277 [10 μg/ml] or both together.

    Techniques: Activation Assay, Activity Assay, Derivative Assay, Concentration Assay, Incubation, Isolation, Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    Blocking the IL-6 signaling pathway results in reduction of the Th17 population stimulated by gingipains . Monocyte-derived dendritic cells were untreated or exposed to P. gingivalis W83 in the presence/absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) for 6 h. Cultures were treated with the inhibitor of JAK1/JAK2 (Ruxolitinib [1 μM]) (A) or antibodies against the IL-6 receptor [10 μg/ml]) (B) for 2 h then naive CD4 + cells were added and cells were co-cultured for 5 or 7 days. At the end of each period, T cells were stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop. For the evaluation of the Th17 population, cells were stained for IL-17A and RORγt, and analyzed by flow cytometry. Data show transcription factor and cytokine positive cells (RORγt + IL-17A + ) and are presented as a mean ± standard deviation of assays performed using three independent donors. Data were analyzed with a Student's t -test ( * P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

    doi: 10.3389/fcimb.2017.00140

    Figure Lengend Snippet: Blocking the IL-6 signaling pathway results in reduction of the Th17 population stimulated by gingipains . Monocyte-derived dendritic cells were untreated or exposed to P. gingivalis W83 in the presence/absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) for 6 h. Cultures were treated with the inhibitor of JAK1/JAK2 (Ruxolitinib [1 μM]) (A) or antibodies against the IL-6 receptor [10 μg/ml]) (B) for 2 h then naive CD4 + cells were added and cells were co-cultured for 5 or 7 days. At the end of each period, T cells were stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop. For the evaluation of the Th17 population, cells were stained for IL-17A and RORγt, and analyzed by flow cytometry. Data show transcription factor and cytokine positive cells (RORγt + IL-17A + ) and are presented as a mean ± standard deviation of assays performed using three independent donors. Data were analyzed with a Student's t -test ( * P

    Article Snippet: Stimulation of moDCS with virulence factors from P. gingivalis Dendritic cells at a concentration of 0.6 × 106 /ml were left untreated (control) or stimulated with Ultra Pure LPS from P. gingivalis (InvivoGen) [2.5 μg/ml], FimA purified from ATCC 33277 [10 μg/ml] or both together.

    Techniques: Blocking Assay, Derivative Assay, Concentration Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Standard Deviation

    Interleukin 6 production induced by P. gingivalis is inversely dependent on gingipain activity . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at 1 μM concentration) or isogenic P. gingivalis gingipain-null mutant ΔKΔRAB for 2 h (A,D,G) , 6 h (B,E,H) , and 24 h ( C , F , I ). (A–C) Supernatants were collected and the concentration of IL-6 was determined by ELISA. (D–F) Arginine-specific and (G–I) Lysine-specific gingipain activity was determined using L-BA p Na and N-( p -Tosyl)-Gly-Pro-Lys-4-nitroanilide acetate salt (200 μM) as a substrate, respectively. Data are presented as means ± standard deviations of assays performed in triplicate using three independent donors, and were analyzed by one-way ANOVA with the Bonferroni post-test correction ( # P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

    doi: 10.3389/fcimb.2017.00140

    Figure Lengend Snippet: Interleukin 6 production induced by P. gingivalis is inversely dependent on gingipain activity . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at 1 μM concentration) or isogenic P. gingivalis gingipain-null mutant ΔKΔRAB for 2 h (A,D,G) , 6 h (B,E,H) , and 24 h ( C , F , I ). (A–C) Supernatants were collected and the concentration of IL-6 was determined by ELISA. (D–F) Arginine-specific and (G–I) Lysine-specific gingipain activity was determined using L-BA p Na and N-( p -Tosyl)-Gly-Pro-Lys-4-nitroanilide acetate salt (200 μM) as a substrate, respectively. Data are presented as means ± standard deviations of assays performed in triplicate using three independent donors, and were analyzed by one-way ANOVA with the Bonferroni post-test correction ( # P

    Article Snippet: Stimulation of moDCS with virulence factors from P. gingivalis Dendritic cells at a concentration of 0.6 × 106 /ml were left untreated (control) or stimulated with Ultra Pure LPS from P. gingivalis (InvivoGen) [2.5 μg/ml], FimA purified from ATCC 33277 [10 μg/ml] or both together.

    Techniques: Activity Assay, Derivative Assay, Concentration Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay

    The role of gingipains in the regulation of cell responses to LPS and fimbriae . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis LPS (2.5 μg/ml), FimA (10 μg/ml) or mixed LPS with FimA. Additionally, cells were co-stimulated with HRgpA (2 nM) in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM). Four hours after stimulation, culture media were collected and cells were lysed with TRIzol. RNA was isolated and reverse transcriptase PCR was performed. Relative expression of the cytokine gene IL-6 to the reference house-keeping gene EF2 was measured by Real-Time PCR. Data show representative fold increase in expression compared to control levels, which were arbitrarily set at 1 (A) . Concentration of IL-6 in collected medium was evaluated by standard ELISA method (B) ; Data are presented as mean ± standard deviation of duplicates from three independent assays and were analyzed with a Student's t -test (# P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

    doi: 10.3389/fcimb.2017.00140

    Figure Lengend Snippet: The role of gingipains in the regulation of cell responses to LPS and fimbriae . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis LPS (2.5 μg/ml), FimA (10 μg/ml) or mixed LPS with FimA. Additionally, cells were co-stimulated with HRgpA (2 nM) in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM). Four hours after stimulation, culture media were collected and cells were lysed with TRIzol. RNA was isolated and reverse transcriptase PCR was performed. Relative expression of the cytokine gene IL-6 to the reference house-keeping gene EF2 was measured by Real-Time PCR. Data show representative fold increase in expression compared to control levels, which were arbitrarily set at 1 (A) . Concentration of IL-6 in collected medium was evaluated by standard ELISA method (B) ; Data are presented as mean ± standard deviation of duplicates from three independent assays and were analyzed with a Student's t -test (# P

    Article Snippet: Stimulation of moDCS with virulence factors from P. gingivalis Dendritic cells at a concentration of 0.6 × 106 /ml were left untreated (control) or stimulated with Ultra Pure LPS from P. gingivalis (InvivoGen) [2.5 μg/ml], FimA purified from ATCC 33277 [10 μg/ml] or both together.

    Techniques: Derivative Assay, Concentration Assay, Isolation, Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Gingipain activity differentially determines the mRNA expression and final secretion of cytokines. (A) Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) or the isogenic gingipain-null mutant ΔKΔRAB at MOI (multiplicity of infection) 1:50. (A) Concentrations of cytokines in collected medium were evaluated by ELISA (IL-6, IL-23, TNF, IFNγ) or by using BD CBA Human Inflammatory Kit (IL-1β, IL-12p70, IL-8, IL-10). A representative result from three separate experiments performed on moDCs derived from different donors is shown. Data are presented as mean ± standard deviation of duplicates from three independent assays and were analyzed with a Student's t -test (# P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

    doi: 10.3389/fcimb.2017.00140

    Figure Lengend Snippet: Gingipain activity differentially determines the mRNA expression and final secretion of cytokines. (A) Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) or the isogenic gingipain-null mutant ΔKΔRAB at MOI (multiplicity of infection) 1:50. (A) Concentrations of cytokines in collected medium were evaluated by ELISA (IL-6, IL-23, TNF, IFNγ) or by using BD CBA Human Inflammatory Kit (IL-1β, IL-12p70, IL-8, IL-10). A representative result from three separate experiments performed on moDCs derived from different donors is shown. Data are presented as mean ± standard deviation of duplicates from three independent assays and were analyzed with a Student's t -test (# P

    Article Snippet: Stimulation of moDCS with virulence factors from P. gingivalis Dendritic cells at a concentration of 0.6 × 106 /ml were left untreated (control) or stimulated with Ultra Pure LPS from P. gingivalis (InvivoGen) [2.5 μg/ml], FimA purified from ATCC 33277 [10 μg/ml] or both together.

    Techniques: Activity Assay, Expressing, Derivative Assay, Concentration Assay, Mutagenesis, Infection, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay, Standard Deviation

    Th17 cells differentiation depends on gingipain activity . Monocyte-derived dendritic cells were untreated or exposed to P. gingivalis W83 in the presence/absence of specific protease inhibitors (KYT-1 and KYT-36, each at the concentration of 1 μM) for 6 h. Naive CD4 + cells were added and co-cultured for an additional 3 or 5 days. At the end of each period, T cells were stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop. For evaluation of the Th17 population, cells were stain for intracellular IL-17A andRORγt, and were analyzed by flow cytometry. Data show a representative dot blot selected from three separate experiments presenting percent of RORγt + , IL-17+, RORγt + IL-17 + after 3 and 5 days of co-stimulation of naive CD4+ T cells with moDC pulsed with P. gingivalis .

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

    doi: 10.3389/fcimb.2017.00140

    Figure Lengend Snippet: Th17 cells differentiation depends on gingipain activity . Monocyte-derived dendritic cells were untreated or exposed to P. gingivalis W83 in the presence/absence of specific protease inhibitors (KYT-1 and KYT-36, each at the concentration of 1 μM) for 6 h. Naive CD4 + cells were added and co-cultured for an additional 3 or 5 days. At the end of each period, T cells were stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop. For evaluation of the Th17 population, cells were stain for intracellular IL-17A andRORγt, and were analyzed by flow cytometry. Data show a representative dot blot selected from three separate experiments presenting percent of RORγt + , IL-17+, RORγt + IL-17 + after 3 and 5 days of co-stimulation of naive CD4+ T cells with moDC pulsed with P. gingivalis .

    Article Snippet: Stimulation of moDCS with virulence factors from P. gingivalis Dendritic cells at a concentration of 0.6 × 106 /ml were left untreated (control) or stimulated with Ultra Pure LPS from P. gingivalis (InvivoGen) [2.5 μg/ml], FimA purified from ATCC 33277 [10 μg/ml] or both together.

    Techniques: Activity Assay, Derivative Assay, Concentration Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Dot Blot

    Expression of interleukin 6 in human monocyte-derived dendritic cells in response to Porphyromonas gingivalis infection . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) or the isogenic gingipain-null mutant ΔKΔRAB for 2 h. After stimulation, cells were lysed with TRIzol, RNA was isolated and reverse transcriptase PCR was performed. Relative expression of IL-6 was measured by using the Real-Time PCR method. A representative qRT-PCR from three separate experiments performed on moDCs derived from different donors is shown. Data are presented as means ± standard deviations of triplicate assays, and were analyzed with the one-way ANOVA with the Bonferroni post-test correction (# P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

    doi: 10.3389/fcimb.2017.00140

    Figure Lengend Snippet: Expression of interleukin 6 in human monocyte-derived dendritic cells in response to Porphyromonas gingivalis infection . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis W83 in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM) or the isogenic gingipain-null mutant ΔKΔRAB for 2 h. After stimulation, cells were lysed with TRIzol, RNA was isolated and reverse transcriptase PCR was performed. Relative expression of IL-6 was measured by using the Real-Time PCR method. A representative qRT-PCR from three separate experiments performed on moDCs derived from different donors is shown. Data are presented as means ± standard deviations of triplicate assays, and were analyzed with the one-way ANOVA with the Bonferroni post-test correction (# P

    Article Snippet: Stimulation of moDCS with virulence factors from P. gingivalis Dendritic cells at a concentration of 0.6 × 106 /ml were left untreated (control) or stimulated with Ultra Pure LPS from P. gingivalis (InvivoGen) [2.5 μg/ml], FimA purified from ATCC 33277 [10 μg/ml] or both together.

    Techniques: Expressing, Derivative Assay, Infection, Concentration Assay, Mutagenesis, Isolation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. gingivalis at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

    doi: 10.3389/fcimb.2017.00359

    Figure Lengend Snippet: TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. gingivalis at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

    Article Snippet: Bacterial growth P. gingivalis (ATCC strains 381 and 53,977 were used in this study) was cultured for 48 h in Wilkins broth (Oxoid, Hampshire, England) without additional nutrients, under anaerobic conditions in Oxoid™ AnaeroJar™ 2.5 L at 37°C.

    Techniques: Blocking Assay, Labeling, Fluorescence, Serial Dilution

    Comparative proteomic of particle-free SN from W50-NIDCR and W50-ATCC. ( A ) Various amounts of particle-free SN from W50-ATCC and W50-NIDCR were resolved by one-dimensional SDS-PAGE and protein bands were visualized by Silver staining. ( B ) Coomassie staining of the same SNs that had been concentrated to 10X as described indicated in the materials and methods. ( C ) Same as (A) except particle-free bacterial SN from W50-ATCC and W50-NIDCR were labeled green or red, respectively, and resolved by two-dimensional difference in-gel electrophoresis.

    Journal: PLoS ONE

    Article Title: Mechanisms by which Porphyromonas gingivalis evades innate immunity

    doi: 10.1371/journal.pone.0182164

    Figure Lengend Snippet: Comparative proteomic of particle-free SN from W50-NIDCR and W50-ATCC. ( A ) Various amounts of particle-free SN from W50-ATCC and W50-NIDCR were resolved by one-dimensional SDS-PAGE and protein bands were visualized by Silver staining. ( B ) Coomassie staining of the same SNs that had been concentrated to 10X as described indicated in the materials and methods. ( C ) Same as (A) except particle-free bacterial SN from W50-ATCC and W50-NIDCR were labeled green or red, respectively, and resolved by two-dimensional difference in-gel electrophoresis.

    Article Snippet: Whole genomic sequencing and comparison of P. gingivalis W50-NIDCR and W50-ATCC The evolutionary adaptation of a microbe to its host often involves multiple genetic modifications.

    Techniques: SDS Page, Silver Staining, Staining, Labeling, Nucleic Acid Electrophoresis

    Mutation in W50-NIDCR HagA causing alteration in the PorSS/gingipain pathway. ( A ) Draft genomes of NCBI W50 and two W50 variant strains (W50-ATCC and W50-NIDCR) were mapped to the reference genome of W83 to illustrate the sequence similarities between these two genomes and the reference. As controls, genomes of P . gingivalis 33277 and E . coli were also included in these analyses. The three outer rings (SNP W50, SNP W50-NIDCR, and SNP W50-ATCC) show the SNP loci indentified by the software “breseq . ” Those that are unique among the three genomes are shown with the +/- affected genes and their annotations in the outer most ring (ORFs) and their annotations. The map was drawn using “BRIG” genome alignment software. ( B ) Red cylindrical domain is a peptidase C25 C-terminal domain, grey cylindrical domains are cleaved adhesin domains, and the red star/arrow indicates the location of W50-NIDCR SNP. Not depicted are the Fibronectin Type III domains, which encompass the cleaved adhesin but extend further in the N- and C-terminal directions. K75* in EIW91729.1 (partial PrtH domain) is analogous to K2074* in AAQ66831.1 (HagA). SNP occurs within the Fibronectin Type III domain at N-terminus of the protein. The raw sequences used in this study have been deposited to the NCBI Short Reads Archive (SRA) under the umbrella BioProject ID PRJNA302472 ( http://www.ncbi.nlm.nih.gov/bioproject/PRJNA302472 ).

    Journal: PLoS ONE

    Article Title: Mechanisms by which Porphyromonas gingivalis evades innate immunity

    doi: 10.1371/journal.pone.0182164

    Figure Lengend Snippet: Mutation in W50-NIDCR HagA causing alteration in the PorSS/gingipain pathway. ( A ) Draft genomes of NCBI W50 and two W50 variant strains (W50-ATCC and W50-NIDCR) were mapped to the reference genome of W83 to illustrate the sequence similarities between these two genomes and the reference. As controls, genomes of P . gingivalis 33277 and E . coli were also included in these analyses. The three outer rings (SNP W50, SNP W50-NIDCR, and SNP W50-ATCC) show the SNP loci indentified by the software “breseq . ” Those that are unique among the three genomes are shown with the +/- affected genes and their annotations in the outer most ring (ORFs) and their annotations. The map was drawn using “BRIG” genome alignment software. ( B ) Red cylindrical domain is a peptidase C25 C-terminal domain, grey cylindrical domains are cleaved adhesin domains, and the red star/arrow indicates the location of W50-NIDCR SNP. Not depicted are the Fibronectin Type III domains, which encompass the cleaved adhesin but extend further in the N- and C-terminal directions. K75* in EIW91729.1 (partial PrtH domain) is analogous to K2074* in AAQ66831.1 (HagA). SNP occurs within the Fibronectin Type III domain at N-terminus of the protein. The raw sequences used in this study have been deposited to the NCBI Short Reads Archive (SRA) under the umbrella BioProject ID PRJNA302472 ( http://www.ncbi.nlm.nih.gov/bioproject/PRJNA302472 ).

    Article Snippet: Whole genomic sequencing and comparison of P. gingivalis W50-NIDCR and W50-ATCC The evolutionary adaptation of a microbe to its host often involves multiple genetic modifications.

    Techniques: Mutagenesis, Variant Assay, Sequencing, Software

    Differential effect of P . gingivalis W50 substrains on extinguishing cytokine production from DCs. (A) DCs from B10.A-Rag2 -/- mice were either unstimulated (medium) or stimulated on the sixth day with 5x10 7 E. coli , F. nucleatum or P. gingivalis alone or a mixture of two bacteria together in a 24-well plate for 16 h at 37°C. CSN were analyzed for the presence of IL-12p75 using a specific ELISA. (B) Same as (A) except that IFNγ was added to the cultures to enhance IL-12 production. P. gingivalis were heat-killed at 60°C for 1 h in a water bath (HKP g) , or not, before being added at 5x10 7 /well to the culture containing F. nucleatum or E. coli . (C) DCs were stimulated with 100 ng/ml of LPS ( E . coli ) plus 100 ng/ml of IFNγ alone (No Pg ) or in combination with various strains of P. gingivalis at 5x10 7 /well for 24 h before testing for the presence of IL-12p75 in the CSN. (D) Same as (C) except various numbers of P. gingivalis W50-NIDCR or -ATCC were added to the DCs in a 24-well plate. (A-D) Data are the mean ± SD of compilation of two independent experiments. (D) The data are expressed as the mean ± SD of duplicates.

    Journal: PLoS ONE

    Article Title: Mechanisms by which Porphyromonas gingivalis evades innate immunity

    doi: 10.1371/journal.pone.0182164

    Figure Lengend Snippet: Differential effect of P . gingivalis W50 substrains on extinguishing cytokine production from DCs. (A) DCs from B10.A-Rag2 -/- mice were either unstimulated (medium) or stimulated on the sixth day with 5x10 7 E. coli , F. nucleatum or P. gingivalis alone or a mixture of two bacteria together in a 24-well plate for 16 h at 37°C. CSN were analyzed for the presence of IL-12p75 using a specific ELISA. (B) Same as (A) except that IFNγ was added to the cultures to enhance IL-12 production. P. gingivalis were heat-killed at 60°C for 1 h in a water bath (HKP g) , or not, before being added at 5x10 7 /well to the culture containing F. nucleatum or E. coli . (C) DCs were stimulated with 100 ng/ml of LPS ( E . coli ) plus 100 ng/ml of IFNγ alone (No Pg ) or in combination with various strains of P. gingivalis at 5x10 7 /well for 24 h before testing for the presence of IL-12p75 in the CSN. (D) Same as (C) except various numbers of P. gingivalis W50-NIDCR or -ATCC were added to the DCs in a 24-well plate. (A-D) Data are the mean ± SD of compilation of two independent experiments. (D) The data are expressed as the mean ± SD of duplicates.

    Article Snippet: Whole genomic sequencing and comparison of P. gingivalis W50-NIDCR and W50-ATCC The evolutionary adaptation of a microbe to its host often involves multiple genetic modifications.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Nearest neighbor ORF of IS 1126 from P. gingivalis ATCC 33277. The open bar represents the total flanking sequence, and the markers are in base pairs. IS 1126 is depicted as the inverted arrowhead, and its sequence (1,338 bp) has been deleted from the schematic. Homologous genes and ORFs are labeled and represented as solid arrows, which are drawn to scale. Also shown is the accession number of each homologous sequence.

    Journal: Infection and Immunity

    Article Title: Genomic Loci of the Porphyromonas gingivalis Insertion Element IS1126

    doi:

    Figure Lengend Snippet: Nearest neighbor ORF of IS 1126 from P. gingivalis ATCC 33277. The open bar represents the total flanking sequence, and the markers are in base pairs. IS 1126 is depicted as the inverted arrowhead, and its sequence (1,338 bp) has been deleted from the schematic. Homologous genes and ORFs are labeled and represented as solid arrows, which are drawn to scale. Also shown is the accession number of each homologous sequence.

    Article Snippet: In the present study, all DNA sequence comparisons were made between the P. gingivalis type strain, ATCC 33277, and strain W83M, obtained from C. Mouton, Laval University, Quebec, Canada.

    Techniques: Sequencing, Labeling

    RFLP analysis of IS 1126 in laboratory strains of P. gingivalis .

    Journal: Infection and Immunity

    Article Title: Genomic Loci of the Porphyromonas gingivalis Insertion Element IS1126

    doi:

    Figure Lengend Snippet: RFLP analysis of IS 1126 in laboratory strains of P. gingivalis .

    Article Snippet: In the present study, all DNA sequence comparisons were made between the P. gingivalis type strain, ATCC 33277, and strain W83M, obtained from C. Mouton, Laval University, Quebec, Canada.

    Techniques:

    Sequences flanking IS 1126 from P. gingivalis ATCC 33277. From each clone, 30-bp flanking sequences are shown 5′ and/or 3′ of IS 1126 , which is depicted centrally in abbreviated form. IR, inverted repeat.

    Journal: Infection and Immunity

    Article Title: Genomic Loci of the Porphyromonas gingivalis Insertion Element IS1126

    doi:

    Figure Lengend Snippet: Sequences flanking IS 1126 from P. gingivalis ATCC 33277. From each clone, 30-bp flanking sequences are shown 5′ and/or 3′ of IS 1126 , which is depicted centrally in abbreviated form. IR, inverted repeat.

    Article Snippet: In the present study, all DNA sequence comparisons were made between the P. gingivalis type strain, ATCC 33277, and strain W83M, obtained from C. Mouton, Laval University, Quebec, Canada.

    Techniques:

    Inhibition of planktonic growth of P. gingivalis by LF-related agents. About 10 4 cells/ml of P. gingivalis JCM 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia ▿

    doi: 10.1128/AAC.01688-08

    Figure Lengend Snippet: Inhibition of planktonic growth of P. gingivalis by LF-related agents. About 10 4 cells/ml of P. gingivalis JCM 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P

    Article Snippet: The level of biofilm formation of P. gingivalis JCM 8525, ATCC 33277, and ATCC 53978 and P. intermedia ATCC 25611 and ATCC 49046 in the presence of several concentrations of native bLF in polyvinylchloride round wells was estimated by measuring the OD550 after staining of the bacteria with crystal violet (Fig. ).

    Techniques: Inhibition, Incubation

    Inhibition of biofilm formation of P. gingivalis and P. intermedia by LF-related agents. About 10 7 cells/ml were incubated in TSB with hemin, vitamin K 1 , and yeast extract in polyvinylchloride plates for 24 h. Biofilm formation was determined by crystal violet staining and is expressed as the OD 550 . P. gingivalis JCM 8525, ATCC 33277, and ATCC 53978 and P. intermedia ATCC 25611 and ATCC 49046 were treated with the indicated concentrations of native bLF (A). P. gingivalis ATCC 33277 (B) and P. intermedia ATCC 25611 (C) were treated with the indicated concentrations of hLF, apo-bLF, native bLF, holo-bLF, or LFcin B. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia ▿

    doi: 10.1128/AAC.01688-08

    Figure Lengend Snippet: Inhibition of biofilm formation of P. gingivalis and P. intermedia by LF-related agents. About 10 7 cells/ml were incubated in TSB with hemin, vitamin K 1 , and yeast extract in polyvinylchloride plates for 24 h. Biofilm formation was determined by crystal violet staining and is expressed as the OD 550 . P. gingivalis JCM 8525, ATCC 33277, and ATCC 53978 and P. intermedia ATCC 25611 and ATCC 49046 were treated with the indicated concentrations of native bLF (A). P. gingivalis ATCC 33277 (B) and P. intermedia ATCC 25611 (C) were treated with the indicated concentrations of hLF, apo-bLF, native bLF, holo-bLF, or LFcin B. Values are means ± SDs ( n = 3) of one representative result from two similar results. *, P

    Article Snippet: The level of biofilm formation of P. gingivalis JCM 8525, ATCC 33277, and ATCC 53978 and P. intermedia ATCC 25611 and ATCC 49046 in the presence of several concentrations of native bLF in polyvinylchloride round wells was estimated by measuring the OD550 after staining of the bacteria with crystal violet (Fig. ).

    Techniques: Inhibition, Incubation, Staining

    Sensitivity of P. gingivalis mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Sensitivity of P. gingivalis mutants to air (A) and air with hydrogen peroxide (B). P. gingivalis cells that had been anaerobically cultured in enriched BHI medium overnight were diluted twice with fresh enriched BHI medium (A) or enriched BHI medium containing hydrogen peroxide (final concentration, 0.5 mM) (B) and then incubated aerobically with vigorous shaking. Samples were withdrawn at intervals and plated, after dilution in enriched BHI medium, on enriched TS plates. The plates were incubated anaerobically at 37°C for 7 days. Symbols: ○, ATCC 33277 (wild type); ▵, KDP139 ( ftn ); •, KDP141 ( dps ); ▴, KDP142 ( ftn dps ).

    Article Snippet: The dps gene DNA disrupted by insertion of the tetQ cartridge DNA was introduced into cells of P. gingivalis wild-type strain (ATCC 33277) and the ftn mutant (KDP139) by electroporation.

    Techniques: Cell Culture, Concentration Assay, Incubation

    Sensitivity of P. gingivalis cells to hydrogen peroxide, mitomycin C, and metronidazole. P. gingivalis ATCC 33277 (wild type), KDP139 ( ftn ), KDP141 ( dps ), and KDP142 ( ftn dps ) were anaerobically grown in enriched BHI medium for 48 h. The cells were spread on enriched TS plates, and a paper disk containing hydrogen peroxide (A), mitomycin C (B), or metronidazole (C) was placed at the centers of the plates, followed by incubation at 37°C anaerobically for 7 days. The diameters of the clear zones next to the disks were measured (in millimeters). The data shown are the means and SD of triplicate experiments.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Sensitivity of P. gingivalis cells to hydrogen peroxide, mitomycin C, and metronidazole. P. gingivalis ATCC 33277 (wild type), KDP139 ( ftn ), KDP141 ( dps ), and KDP142 ( ftn dps ) were anaerobically grown in enriched BHI medium for 48 h. The cells were spread on enriched TS plates, and a paper disk containing hydrogen peroxide (A), mitomycin C (B), or metronidazole (C) was placed at the centers of the plates, followed by incubation at 37°C anaerobically for 7 days. The diameters of the clear zones next to the disks were measured (in millimeters). The data shown are the means and SD of triplicate experiments.

    Article Snippet: The dps gene DNA disrupted by insertion of the tetQ cartridge DNA was introduced into cells of P. gingivalis wild-type strain (ATCC 33277) and the ftn mutant (KDP139) by electroporation.

    Techniques: Incubation

    DNA-binding activity of P. gingivalis Dps. Recombinant P. gingivalis Dps purified from the E. coli overexpressing P. gingivalis dps or the recombinant P. gingivalis Dps treated with ferrous ammonium sulfate was incubated with linear DNA (1-kb DNA ladder) at 4°C for 1 h. The mixture was then subjected to agarose gel electrophoresis. DNA on the gel was stained with ethidium bromide. Lanes: 1, DNA (500 ng) alone; 2, recombinant Dps (10 μg) alone; 3, recombinant Dps (10 μg) and DNA (500 ng); 4, iron-loaded recombinant Dps (10 μg) and DNA (500 ng); 5, iron-loaded recombinant Dps (10 μg) alone.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: DNA-binding activity of P. gingivalis Dps. Recombinant P. gingivalis Dps purified from the E. coli overexpressing P. gingivalis dps or the recombinant P. gingivalis Dps treated with ferrous ammonium sulfate was incubated with linear DNA (1-kb DNA ladder) at 4°C for 1 h. The mixture was then subjected to agarose gel electrophoresis. DNA on the gel was stained with ethidium bromide. Lanes: 1, DNA (500 ng) alone; 2, recombinant Dps (10 μg) alone; 3, recombinant Dps (10 μg) and DNA (500 ng); 4, iron-loaded recombinant Dps (10 μg) and DNA (500 ng); 5, iron-loaded recombinant Dps (10 μg) alone.

    Article Snippet: The dps gene DNA disrupted by insertion of the tetQ cartridge DNA was introduced into cells of P. gingivalis wild-type strain (ATCC 33277) and the ftn mutant (KDP139) by electroporation.

    Techniques: Binding Assay, Activity Assay, Recombinant, Purification, Incubation, Agarose Gel Electrophoresis, Staining

    Alignment of the amino acid sequence of P. gingivalis Dps with those of the Dps previously isolated and determined in other prokaryotes.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Alignment of the amino acid sequence of P. gingivalis Dps with those of the Dps previously isolated and determined in other prokaryotes.

    Article Snippet: The dps gene DNA disrupted by insertion of the tetQ cartridge DNA was introduced into cells of P. gingivalis wild-type strain (ATCC 33277) and the ftn mutant (KDP139) by electroporation.

    Techniques: Sequencing, Isolation

    Time course of induction of β-galactosidase activity in the dps′-′lacZ fusion strains. P. gingivalis KDP146 ( dps + dps′-′lacZ ) (circles) and KDP148 ( oxyR dps + dps′-′lacZ ) (triangles) were grown anaerobically in enriched BHI medium at 37°C. At an A 600 ). The β-galactosidase activity of the wild-type parent strain ATCC 33277 was

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Time course of induction of β-galactosidase activity in the dps′-′lacZ fusion strains. P. gingivalis KDP146 ( dps + dps′-′lacZ ) (circles) and KDP148 ( oxyR dps + dps′-′lacZ ) (triangles) were grown anaerobically in enriched BHI medium at 37°C. At an A 600 ). The β-galactosidase activity of the wild-type parent strain ATCC 33277 was

    Article Snippet: The dps gene DNA disrupted by insertion of the tetQ cartridge DNA was introduced into cells of P. gingivalis wild-type strain (ATCC 33277) and the ftn mutant (KDP139) by electroporation.

    Techniques: Activity Assay

    Proof of authenticity of the P. gingivalis dps mutant KDP141 and the ftn dps double mutant KDP142. (A and B) Southern blot analyses of the chromosomal DNA. The chromosomal DNAs of the wild-type ATCC 33277 (lane 1) and the ftn mutants KDP139 (lane 2), KDP141 (lane 3), and KDP142 (lane 4) were digested with Nco I. The resulting DNA fragments were subjected to agarose gel electrophresis, followed by blotting. Hybridization was performed by using the 0.6-kb Nde I- Bgl II fragment of pKD390 as a dps probe (A) and the 2.7-kb Bam HI- Bgl II fragment of pKD375 as a tetQ probe (B). (C) Immunoblot analysis. After purified P. gingivalis Dps (lane 1) and the cell extracts of ATCC 33277 (lane 2), KDP141 (lane 3), and KDP142 (lane 4) were electrophoresed through an SDS-polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane and immunoreacted with antiserum against P. gingivalis Dps.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Proof of authenticity of the P. gingivalis dps mutant KDP141 and the ftn dps double mutant KDP142. (A and B) Southern blot analyses of the chromosomal DNA. The chromosomal DNAs of the wild-type ATCC 33277 (lane 1) and the ftn mutants KDP139 (lane 2), KDP141 (lane 3), and KDP142 (lane 4) were digested with Nco I. The resulting DNA fragments were subjected to agarose gel electrophresis, followed by blotting. Hybridization was performed by using the 0.6-kb Nde I- Bgl II fragment of pKD390 as a dps probe (A) and the 2.7-kb Bam HI- Bgl II fragment of pKD375 as a tetQ probe (B). (C) Immunoblot analysis. After purified P. gingivalis Dps (lane 1) and the cell extracts of ATCC 33277 (lane 2), KDP141 (lane 3), and KDP142 (lane 4) were electrophoresed through an SDS-polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane and immunoreacted with antiserum against P. gingivalis Dps.

    Article Snippet: The dps gene DNA disrupted by insertion of the tetQ cartridge DNA was introduced into cells of P. gingivalis wild-type strain (ATCC 33277) and the ftn mutant (KDP139) by electroporation.

    Techniques: Mutagenesis, Southern Blot, Agarose Gel Electrophoresis, Hybridization, Purification

    Survival of P. gingivalis cells in HUVEC. Monolayers of HUVEC were infected by P. gingivalis ATCC 33277 (wild type) and KDP141 ( dps ). Experiments were done at least five times, and the data are presented as the mean and the SD of the CFU per HUVEC.

    Journal: Infection and Immunity

    Article Title: Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe Porphyromonas gingivalis

    doi: 10.1128/IAI.71.3.1170-1178.2003

    Figure Lengend Snippet: Survival of P. gingivalis cells in HUVEC. Monolayers of HUVEC were infected by P. gingivalis ATCC 33277 (wild type) and KDP141 ( dps ). Experiments were done at least five times, and the data are presented as the mean and the SD of the CFU per HUVEC.

    Article Snippet: The dps gene DNA disrupted by insertion of the tetQ cartridge DNA was introduced into cells of P. gingivalis wild-type strain (ATCC 33277) and the ftn mutant (KDP139) by electroporation.

    Techniques: Infection

    Cleavage (A) and activation (B) of procaspase-3 by P. gingivalis -excreted gingipains. (A) Incubation of pure procaspase-3 with caspase-8 (4 or 8 ng) or culture supernatants ('sup') of wild type (WT) or triple gingipain mutant (MT) P. gingivalis (20 or 40 ng protein content, labeled over lanes) were done as described under Experimental Procedures. The products were analyzed by SDS-PAGE and silver-staining. The full-length procaspase-3 (34 kDa) and the processed active fragments (17 and 12 kDa) are marked by open and closed arrowheads, respectively. (B) Activity of the processed caspase-3 generated in Panel A was measured using a colorimetric peptide substrate described under Experimental Procedures. The enzymes used to cleave procsapase-3 are: 20 ng of wild type P. gingivalis sup (Triangle); 4 ng caspase-8 (Squares); 20 ng of gingipain mutant P. gingivalis sup (Open circles); 20 ng of wild type P. gingivalis sup, preheated at 65°C for 15 min (Diamonds); none (Closed circles). The error bars are derived from three independent experiments.

    Journal: BMC Microbiology

    Article Title: Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling

    doi: 10.1186/1471-2180-6-26

    Figure Lengend Snippet: Cleavage (A) and activation (B) of procaspase-3 by P. gingivalis -excreted gingipains. (A) Incubation of pure procaspase-3 with caspase-8 (4 or 8 ng) or culture supernatants ('sup') of wild type (WT) or triple gingipain mutant (MT) P. gingivalis (20 or 40 ng protein content, labeled over lanes) were done as described under Experimental Procedures. The products were analyzed by SDS-PAGE and silver-staining. The full-length procaspase-3 (34 kDa) and the processed active fragments (17 and 12 kDa) are marked by open and closed arrowheads, respectively. (B) Activity of the processed caspase-3 generated in Panel A was measured using a colorimetric peptide substrate described under Experimental Procedures. The enzymes used to cleave procsapase-3 are: 20 ng of wild type P. gingivalis sup (Triangle); 4 ng caspase-8 (Squares); 20 ng of gingipain mutant P. gingivalis sup (Open circles); 20 ng of wild type P. gingivalis sup, preheated at 65°C for 15 min (Diamonds); none (Closed circles). The error bars are derived from three independent experiments.

    Article Snippet: Bacteria and culture conditions Wild-type P. gingivalis (ATCC 33277) and the isogenic mutant strain (rgpA rgpB kgp ) [ ] were grown anaerobically (85% N2 , 10% H2 , and 5% CO2 ) at 37°C in brain-heart infusion broth supplemented with yeast extract (0.5%), hemin (5 μg/ml), and menadione (0.5 μg/ml), essentially as described [ ].

    Techniques: Activation Assay, Incubation, Mutagenesis, Labeling, SDS Page, Silver Staining, Activity Assay, Generated, Derivative Assay

    Induction of apoptosis-related gene mRNAs in HGF cells infected with wild type P. gingivalis (Triangles) or its triple gingipain mutant (Circles). Total mRNA from infected cells at different times p.i. were subjected to quantitative Real Time RT-PCR as described under Materials and Methods, using primers described in Table 1. The relative amounts of RNA were expressed as the ratio of uninfected control value (fold induction). Each box represents a specific gene as named and its generally accepted nature as anti-apoptotic ('anti') or pro-apoptotic ('pro'). Each data point is derived from three independent infection experiments with the error bar as shown. Note the general trend of early activation of anti-apoptotic genes (upper rows) and late activation of the pro-apoptotic ones (lower rows). Treatment with LY294002 (Closed circles) and SN50 (Closed triangles) inhibited activation of anti-apoptotic genes, as exemplified by Bfl-1.

    Journal: BMC Microbiology

    Article Title: Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling

    doi: 10.1186/1471-2180-6-26

    Figure Lengend Snippet: Induction of apoptosis-related gene mRNAs in HGF cells infected with wild type P. gingivalis (Triangles) or its triple gingipain mutant (Circles). Total mRNA from infected cells at different times p.i. were subjected to quantitative Real Time RT-PCR as described under Materials and Methods, using primers described in Table 1. The relative amounts of RNA were expressed as the ratio of uninfected control value (fold induction). Each box represents a specific gene as named and its generally accepted nature as anti-apoptotic ('anti') or pro-apoptotic ('pro'). Each data point is derived from three independent infection experiments with the error bar as shown. Note the general trend of early activation of anti-apoptotic genes (upper rows) and late activation of the pro-apoptotic ones (lower rows). Treatment with LY294002 (Closed circles) and SN50 (Closed triangles) inhibited activation of anti-apoptotic genes, as exemplified by Bfl-1.

    Article Snippet: Bacteria and culture conditions Wild-type P. gingivalis (ATCC 33277) and the isogenic mutant strain (rgpA rgpB kgp ) [ ] were grown anaerobically (85% N2 , 10% H2 , and 5% CO2 ) at 37°C in brain-heart infusion broth supplemented with yeast extract (0.5%), hemin (5 μg/ml), and menadione (0.5 μg/ml), essentially as described [ ].

    Techniques: Infection, Mutagenesis, Quantitative RT-PCR, Derivative Assay, Activation Assay

    Activation of members of the PI3K/AKT pathway. HGF monolayer was infected with wild type P. gingivalis (WT) or its triple gingipain mutant (MT), and the infected cells grown in the presence (+I) or absence of 20 μM LY294002 (PI3K inhibitor). Total cell extracts (50 μg) were probed in immunoblot for the presence of total AKT or specific phosphorylated proteins of the AKT pathway as named. The two species of phospho-FKHR are indicated by closed circle and triangle. Note the early (6–12 h) activation of phosphorylation and its inhibition by the inhibitor.

    Journal: BMC Microbiology

    Article Title: Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling

    doi: 10.1186/1471-2180-6-26

    Figure Lengend Snippet: Activation of members of the PI3K/AKT pathway. HGF monolayer was infected with wild type P. gingivalis (WT) or its triple gingipain mutant (MT), and the infected cells grown in the presence (+I) or absence of 20 μM LY294002 (PI3K inhibitor). Total cell extracts (50 μg) were probed in immunoblot for the presence of total AKT or specific phosphorylated proteins of the AKT pathway as named. The two species of phospho-FKHR are indicated by closed circle and triangle. Note the early (6–12 h) activation of phosphorylation and its inhibition by the inhibitor.

    Article Snippet: Bacteria and culture conditions Wild-type P. gingivalis (ATCC 33277) and the isogenic mutant strain (rgpA rgpB kgp ) [ ] were grown anaerobically (85% N2 , 10% H2 , and 5% CO2 ) at 37°C in brain-heart infusion broth supplemented with yeast extract (0.5%), hemin (5 μg/ml), and menadione (0.5 μg/ml), essentially as described [ ].

    Techniques: Activation Assay, Infection, Mutagenesis, Inhibition

    PI3K-dependent activation of NF-κB by P. gingivalis . (A) Activation of NF-κB-dependent luciferase in HEK cells. Monolayers were infected with wild type P. gingivalis in the presence of no drug (Open bars), 20 μM SN50 (Speckled bars), 20 μM LY294002 (Striped bar), or with the triple gingipain mutant (Closed bars), and luciferase assay carried out as described in Materials and Methods. Error bars represent average of three experiments. (B) Nuclear import of NF-κB p65 subunit. HGF cells were infected with P. gingivalis (wild type = WT; gingipain mutant = MT), and nuclear extracts (40 μg protein) of infected cells (or uninfected controls = U) were analyzed for p65 by immunoblot [54]. Sp1 serves as an unchanged control. (C) Phosphorylated IκB-α, an indicator of NF-κB activation, was detected by immunoblot of the total infected HGF cell extract using a specific antibody [54]. This phosphorylation was undetectable when 20 μM LY294002 (PI3K inhibitor) was included in the culture medium (+I).

    Journal: BMC Microbiology

    Article Title: Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling

    doi: 10.1186/1471-2180-6-26

    Figure Lengend Snippet: PI3K-dependent activation of NF-κB by P. gingivalis . (A) Activation of NF-κB-dependent luciferase in HEK cells. Monolayers were infected with wild type P. gingivalis in the presence of no drug (Open bars), 20 μM SN50 (Speckled bars), 20 μM LY294002 (Striped bar), or with the triple gingipain mutant (Closed bars), and luciferase assay carried out as described in Materials and Methods. Error bars represent average of three experiments. (B) Nuclear import of NF-κB p65 subunit. HGF cells were infected with P. gingivalis (wild type = WT; gingipain mutant = MT), and nuclear extracts (40 μg protein) of infected cells (or uninfected controls = U) were analyzed for p65 by immunoblot [54]. Sp1 serves as an unchanged control. (C) Phosphorylated IκB-α, an indicator of NF-κB activation, was detected by immunoblot of the total infected HGF cell extract using a specific antibody [54]. This phosphorylation was undetectable when 20 μM LY294002 (PI3K inhibitor) was included in the culture medium (+I).

    Article Snippet: Bacteria and culture conditions Wild-type P. gingivalis (ATCC 33277) and the isogenic mutant strain (rgpA rgpB kgp ) [ ] were grown anaerobically (85% N2 , 10% H2 , and 5% CO2 ) at 37°C in brain-heart infusion broth supplemented with yeast extract (0.5%), hemin (5 μg/ml), and menadione (0.5 μg/ml), essentially as described [ ].

    Techniques: Activation Assay, Luciferase, Infection, Mutagenesis