Journal: European Journal of Histochemistry : EJH

Article Title: Histochemical localization of sialic acids and antimicrobial substances in eccrine glands of porcine snout skin

doi: 10.4081/ejh.2012.e6

Figure Lengend Snippet: mmunohistochemical staining in the eccrine glands of the snout skin. a) Lysozyme, arrow: dark cell; b) IgA, C: capillary, arrow: dark cell; c) lactoferrin, arrow: dark cell; d) β-defensin 2, arrow: dark cell; e) Rab3D, arrow: dark cell; f) control.

Article Snippet: Then, they were incubated with primary antibodies diluted in 0.01 M PBS (pH 7.3) against lysozyme (polyclonal, from rabbit) (Dako, Glostrup, Denmark), IgA (polyclonal, from rabbit) (Dako), lactoferrin (polyclonal, from rabbit) (MP Biomedicals, Ohio, USA), human β-defensin 2 (polyclonal, from rabbit) (Biolog, Kronshagen, Germany) and Rab3D (polyclonal, from rabbit) (Proteintech, Illinois, USA) for 24 h at 4°C, following preincubation with 1% BSA in PBS for 30 min at room temperature.

Techniques: Staining

Journal: Infection and Immunity

Article Title: Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

doi: 10.1128/IAI.00875-17

Figure Lengend Snippet: The antimicrobial activity of hBD1ox is specific for Gram-negative bacteria. HBD1ox and hBD1red were incubated with Gram-negative bacteria in an aerobic (A) or anaerobic (B) environment in the radial diffusion assay. Antimicrobial activity was measured by analyzing the diameter of the inhibition zone. A diameter of 2.5 mm (dotted line) is the diameter of the punched well. Data are presented as mean ± SEM from three independent experiments.

Article Snippet: Oxidized hBD1 (hBD1ox) was ordered from Peptide Institute, Japan.

Techniques: Activity Assay, Incubation, Diffusion-based Assay, Inhibition

Journal: Infection and Immunity

Article Title: Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

doi: 10.1128/IAI.00875-17

Figure Lengend Snippet: Localization of hBD1ox in bacterial compartments. WT E. coli MC1000 and E. coli MC1000 ΔdsbA ΔdsbB were treated with hBD1ox for 2 h. The samples were fixed and incubated with antibodies against the hBD1ox. The secondary antibodies were conjugated to 6-nm gold particles (black points) and visualized by electron microscopy. (A) Electron microscopy pictures from one representative experiment. Scale bar, 0.2 μm. (B) Visual analysis of electron microscopy pictures. Gold-labeled hBD1ox was counted in bacterial periplasm or cytosol per bacterium. Gold particles per bacterium (percent) are represented (WT, n = 39; ΔdsbA ΔdsbB mutant, n = 30) as determined by four independent individuals.

Article Snippet: Oxidized hBD1 (hBD1ox) was ordered from Peptide Institute, Japan.

Techniques: Incubation, Electron Microscopy, Labeling, Mutagenesis

Journal: Infection and Immunity

Article Title: Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

doi: 10.1128/IAI.00875-17

Figure Lengend Snippet: Antimicrobial activity against bacteria with mutations in the genes of interest. (A) Schematic overview of located membrane and periplasmic proteins in bacteria. (B to D) Two micrograms of hBD1ox and hBD1red was tested against E. coli strains with different protein knockouts in the outer membrane (B), cytosol (C), and periplasmic space (D) by radial diffusion assay. (D) HBD1ox shows a decreased antimicrobial activity against bacteria without DsbA or inner membrane proteins DsbB and TonB in comparison to the WT E. coli MC1000. The diameter of the inhibition zone was measured in the radial diffusion assay to determine the antimicrobial activity. A diameter of 2.5 mm (dotted line) is the diameter of the punched well. Results from experiments with wild-type E. coli MC1000 were pooled (n = 18) and used as the control for all the times when E. coli MC1000 mutants (n = 3) are assessed. All data are presented as mean ± SEM.

Article Snippet: Oxidized hBD1 (hBD1ox) was ordered from Peptide Institute, Japan.

Techniques: Activity Assay, Diffusion-based Assay, Inhibition

Journal: Infection and Immunity

Article Title: Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

doi: 10.1128/IAI.00875-17

Figure Lengend Snippet: Bacterial oxidoreductases DsbA and DsbB are essential for the activity of hBD1ox. (A) Two micrograms of hBD1ox and hBD1red was tested against E. coli with protein double knockouts in the redox system and outer membrane. Bacteria without both oxidoreductases obtain resistance against hBD1ox (**, P = 0.0044; ****, P < 0.0001). (B) Membrane depolarization assay of E. coli MC1000 ΔdsbA ΔdsbB incubated with reduced and oxidized hBD1 for 1 h. HBD3ox (50 μg/ml) was used as positive control (+), and the negative control (−) was incubated without any peptide. HBD1ox is not able to significantly affect the bacterial membrane (**, P = 0.0067). (C) Four micrograms of hBD1ox was used in the plasmid induction experiment with either E. coli MC1000 ΔdsbA ΔdsbB, E. coli MC1000 ΔdsbA ΔdsbB harboring plasmids pQE60::dsbB and pBAD33::dsbA to generate the wild-type phenotype, or, as a control, E. coli MC1000 ΔdsbA ΔdsbB with empty plasmids pQE60 and pBAD33. Plasmid expression was during the 2.5 h of incubation, with addition of 0.4% l-arabinose and 2 mM IPTG every 30 min. HBD1ox shows antimicrobial activity against the mutant containing plasmids pQE60::dsbB and pBAD33::dsbA (**, P = 0.0036; ****, P < 0.0001). The diameter of the inhibition zone was measured in a radial diffusion assay to determine the antimicrobial activity. Results of representative radial diffusion assays are shown. A diameter of 2.5 mm (dotted line) is the diameter of the punched well. Data are presented as mean ± SEM from at least three independent biological replicates. Student's t test was used for comparison of normally distributed data.

Article Snippet: Oxidized hBD1 (hBD1ox) was ordered from Peptide Institute, Japan.

Techniques: Activity Assay, Incubation, Positive Control, Negative Control, Plasmid Preparation, Expressing, Mutagenesis, Inhibition, Diffusion-based Assay

Journal: Infection and Immunity

Article Title: Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

doi: 10.1128/IAI.00875-17

Figure Lengend Snippet: HBD1ox induces bleb formation in E. coli. (A) WT E. coli MC1000 and E. coli MC1000 ΔdsbA ΔdsbB were treated with hBD1ox for 2 h. The samples were fixed in Karnovsky's reagent, and the morphology was analyzed with scanning electron microscopy. Electron microscopy pictures from one representative experiment are shown. Scale bar, 2 μm. (B) Visual analysis of electron microscopy pictures. Outer membrane vesicles (blebs) were counted and determined by four different experts. Data are presented as mean ± SEM from the analyzed bacteria (n = 10). The statistical significance was evaluated by using Student's t test (**, P = 0.0031).

Article Snippet: Oxidized hBD1 (hBD1ox) was ordered from Peptide Institute, Japan.

Techniques: Electron Microscopy

Journal: Infection and Immunity

Article Title: Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

doi: 10.1128/IAI.00875-17

Figure Lengend Snippet: Schematic model describing a potential mechanism of hBD1ox activity. (A) Proposed model for cell death in case of an intact DsbA/DsbB complex. HBD1ox can diffuse in the periplasm by using FepA or FhuA, where it can interact directly or indirectly with DsbA/DsbB. Subsequently, the accumulation of hBD1ox in the periplasm induces bleb formation and finally bacterial cell lysis by an unknown mechanism. (B) Hypothetical model for hBD1ox resistance in bacteria without a traditional DsbA/DsbB complex. Without DsbA/DsbB, hBD1ox diffuses into the cytosol and is finally degraded.

Article Snippet: Oxidized hBD1 (hBD1ox) was ordered from Peptide Institute, Japan.

Techniques: Activity Assay, Lysis

Journal: Pathogens

Article Title: Identification of a Neisseria gonorrhoeae Histone Deacetylase: Epigenetic Impact on Host Gene Expression

doi: 10.3390/pathogens9020132

Figure Lengend Snippet: Gonococcal infection downregulates LL-37 and HBD-1 expression in monocytes. ( A ): Antimicrobial host defense peptide (AMP) gene expression in THP-1 cells infected with Gc strain FA19 overnight at multiplicity of infection (MOI) of 1, 5 and 25 measured by qRT-PCR. ( B ): LL-37 gene expression in primary human monocytes infected with Gc strain FA19 overnight at MOI of 10 measured by qRT-PCR. Peripheral monocytes were obtained from four different healthy donors. ( C ): Overexpression of LL-37 gene in human THP-1 monocytes treated with 10 nM of 1,25 dihydroxyvitamin D3 (VitD3) overnight prior to infection with Gc strain FA19 at MOI of 25. Downregulation of LL-37 gene expression was assessed at 5h and 18h post-infection. p values were calculated using a Student’s t -test in reference to noninfected cells (**). p values in reference to infection at MOI 1 for LL-37 expression (*), HBD1 (#) and SLPI ($). These data are representative of three independent experiments.

Article Snippet: The following primer sets (Quantitect ® Primer Assay) were purchased from Qiagen: human HBD-1 (Hs_DEFB_1_SG), human SLPI (Hs_SLPI_1_SG), human HBD-5 (Hs_DEFB105A_3_SG) and hHDAC1 (Hs_HDAC1_1_SG).

Techniques: Infection, Expressing, Quantitative RT-PCR, Over Expression

Journal: Pathogens

Article Title: Identification of a Neisseria gonorrhoeae Histone Deacetylase: Epigenetic Impact on Host Gene Expression

doi: 10.3390/pathogens9020132

Figure Lengend Snippet: Expression of Neisseria gonorrhoeae AMPs gene expression in infected monocytes in the presence and absence of Gc-HDAC. ( A ): Expression of LL-37, HBD-1 and SLPI genes in THP-1 monocytes infected with WT strain FA19 or its isogenic Gc-HDAC-deficient mutant or the complemented H’C strain at MOI of 1 overnight (n = 3) was assessed using qRT-PCR. ( B ): H3K9ac epigenetic mark enrichment at the promoters of LL-37 and HBD-1 in THP-1 monocytes infected with WT strain FA19 or its isogenic Gc-HDAC-deficient mutant at MOI of 25 overnight (n = 3) was assessed using a ChIP assay. p values were calculated using a Student’s t -test in reference to cells infected with the WT FA19 strain for LL-37 expression (*), HBD1 (#) and SLPI ($).

Article Snippet: The following primer sets (Quantitect ® Primer Assay) were purchased from Qiagen: human HBD-1 (Hs_DEFB_1_SG), human SLPI (Hs_SLPI_1_SG), human HBD-5 (Hs_DEFB105A_3_SG) and hHDAC1 (Hs_HDAC1_1_SG).

Techniques: Expressing, Infection, Mutagenesis, Quantitative RT-PCR