Journal:

Article Title: RhuR, an Extracytoplasmic Function Sigma Factor Activator, Is Essential for Heme-Dependent Expression of the Outer Membrane Heme and Hemoprotein Receptor of Bordetella avium

doi: 10.1128/IAI.72.2.896-907.2004

Figure Lengend Snippet: The N terminus of RhuR activates PbhuR in the absence of hemin. Fe-stressed overnight cultures were used to inoculate high-Fe (BHI plus 150 μM FeSO4), low-Fe (BHI plus 100 μM EDDHA), and low-Fe plus hemin (BHI plus 100 μM EDDHA plus 1 μM hemin) broth cultures. After 16 h of incubation, the β-galactosidase activity of the cultures was determined using a modified Miller assay (27). (A) Activity of the RhuR fusions in wt B. avium. pAEK21 expresses a chimeric protein consisting of the first 97 amino acids of RhuR fused to β-lactamase (BlaM); pAEK23 encodes the same 97-amino-acid region of RhuR fused to 18 irrelevant amino acids encoded by the vector. (B) PbhuR promoter activity of the RhuR fusions in 4169rifΔrhuR. Numbers represent the average β-galactosidase activity of triplicate cultures. Error bars indicate ±1 standard deviation. Asterisks indicate a significant difference (P < 0.05) from the vector control cultured under the same condition, as determined by Student's t test.

Article Snippet: Rabbit polyclonal anti-β-lactamase antibodies (5 Prime-3 Prime, Inc., Boulder, Colo.) and peroxidase-conjugated anti-rabbit immunoglobulin G (whole molecule; Sigma Biochemicals) antibodies were used to detect RhuR-BlaM fusion protein.

Techniques: Incubation, Activity Assay, Modification, Plasmid Preparation, Standard Deviation, Cell Culture

Journal:

Article Title: RhuR, an Extracytoplasmic Function Sigma Factor Activator, Is Essential for Heme-Dependent Expression of the Outer Membrane Heme and Hemoprotein Receptor of Bordetella avium

doi: 10.1128/IAI.72.2.896-907.2004

Figure Lengend Snippet: RhuR-BlaM is localized to the inner membrane of 4169rifΔrhuR. Fe-stressed cultures of 4169rifΔrhuR(pDJM41, pRK415) (vector) and 4169rifΔrhuR(pDJM41, pAEK21) (RhuR-BlaM) were used to inoculate BHI broth supplemented with 100 μM EDDHA and 1 μM hemin. At stationary phase, cells were collected for isolation of total membranes, outer membranes, and cytoplasmic membranes. Ten micrograms of total protein from each membrane preparation was separated by SDS-PAGE using SDS-12% polyacrylamide gels. Western immunoblotting using a rabbit polyclonal anti-BlaM antiserum was used to detect immunoreactive proteins. The positions of the molecular mass markers (in kilodaltons) are indicated. Arrows indicate the position of the 37-kDa RhuR-BlaM and its major 25-kDa degradation product.

Article Snippet: Rabbit polyclonal anti-β-lactamase antibodies (5 Prime-3 Prime, Inc., Boulder, Colo.) and peroxidase-conjugated anti-rabbit immunoglobulin G (whole molecule; Sigma Biochemicals) antibodies were used to detect RhuR-BlaM fusion protein.

Techniques: Plasmid Preparation, Isolation, SDS Page, Western Blot

Journal: PLOS Pathogens

Article Title: Mlf mediates proteotoxic response via formation of cellular foci for protein folding and degradation in Giardia

doi: 10.1371/journal.ppat.1012617

Figure Lengend Snippet: (A) Localization of endogenously BAP-tagged GiMlf in Giardia using confocal microscopy. The cells were stained with an anti-BAP antibody (green), an anti-GL50803_9296 antibody (mitosomal marker; magenta), nuclei stained with DAPI (blue). DIC image of the corresponding cell is shown in the corner of the merged image, scale bar: 2 μm. (B,C) Localization of GiMlf in trophozoites using expansion microscopy (ExM), 3.7 expansion factor. Scale bars: 4 μm. (B) The cells were stained with an anti-GiMlf antibody (green) and an anti-acetylated tubulin antibody (magenta) or (C) anti-BAP antibody (green) and NHS ester dye that labels primary amines of proteins. The basal bodies are marked by asterisks, the dense band is indicated by an arrowhead and the disc margin is marked by an arrow. (D) Localization of GiMlf in encysting cells (48 h post induction) using confocal microscopy. The cells were stained with anti-CWP1 antibody (green) and anti-BAP antibody (magenta), nuclei stained with DAPI (blue). The disc margin is marked by an arrow. The DIC image of the corresponding cell is shown in the corner of the merged image, scale bar: 2 μm. (E) Electron tomography of the central region between two Giardia nuclei depicting immunogold-labeled endogenously V5-tagged GiMlf and reconstructed subcellular structures. The image shows the presence of GiMlf (magenta dots) at the base of the basal bodies (green), adhesive disc microtubules (yellow) and at the mitosomes (red), scale bar: 250 nm. (F) Western blot of Giardia cellular fractions labeled with anti-GiMlf antibody and compartment-specific antibodies. Lysate (lys), low-speed pellet (LSP; containing cytoskeleton and nuclei), cytoplasm (cyto) and high-speed pellet (HSP; containing membrane-bound organelles), 9296—mitosomal marker protein (GL50803_9296) of unknown function, PDI2 –ER marker–protein disulfide isomerase 2, enolase–cytosolic marker, ac. tubulin–acetylated tubulin. (G) Trypsin treatment of the combined LSP and HSP fractions in the presence or absence of 1% Triton X-100. Tom40 and GL50803_9296 were used as markers for protease-accessible and membrane-protected proteins, respectively. (H) The combined fractions of LSP and HSP were treated by sodium carbonate to release peripherally associated proteins from cellular membranes. S–fraction of proteins released into the supernatant, P–fraction of proteins pelleted together with membranes. Sec20, VAP, and PDI2 were used as markers for integral membrane proteins. (I) The membrane lipid strip assay demonstrates the affinity of GiMlf for signaling components within the membrane. Recombinant GiMlf was detected using an anti-GiMlf antibody. TG–triglyceride, DAG–diacylglycerol, PA–phosphatidic acid, PS–phosphatidylserine, PE–phosphatidylethanolamine, PC–phosphatidylcholine, PG–phosphatidylglycerol, CL–cardiolipin, PI–phosphatidylinositol, C–cholesterol, SM–sphingomyelin, 3-SGC– 3-sulfogalactosylceramide, PI(4)P–phosphatidylinositol (4)-phosphate, PI(4,5)P 2 –phosphatidylinositol (4,5)-bisphosphate, PI(3,4,5)P 3 –phosphatidylinositol (3,4,5)-trisphosphate.

Article Snippet: For the immunolabeling, commercial primary antibodies mouse anti-BAP (1:500, GenScript), rabbit anti-V5 (1:500, Abcam), mouse anti-acetylated tubulin (1:1000, Sigma-Aldrich–T7451), self-made primary antibodies rabbit anti-Tom40 (1:1000, [ ], rat anti-PDI2 (1:1000, [ ], rat anti-GiMlf (1:500, [ ] and commercial secondary anti-rabbit/mouse Alexa Fluor 647, anti-rat/mouse Alexa Fluor 488, and anti-rabbit Alexa Fluor 594 (1:500, Invitrogen) antibodies were used.

Techniques: Confocal Microscopy, Staining, Marker, Microscopy, Tomography, Labeling, Adhesive, Western Blot, Membrane, Stripping Membranes, Recombinant

Journal: PLOS Pathogens

Article Title: Mlf mediates proteotoxic response via formation of cellular foci for protein folding and degradation in Giardia

doi: 10.1371/journal.ppat.1012617

Figure Lengend Snippet: (A) Volcano plot of the isolated GiMlf interactome using DSP crosslinking (P≤0.02, fold change >2, n = 3). Cytoskeletal components, PQC proteins, and transmembrane proteins are highlighted as indicated in the legend. Transmembrane topology prediction was performed using the DeepTMHMM tool. The indicated proteins were further analyzed in this study. (B) Localization of endogenously BAP-tagged GiMlf and V5-tagged putative interacting partners in trophozoites of G . intestinalis using confocal microscopy. The cells were stained with anti-BAP antibody (green) and anti-V5 antibody (magenta), scale bar: 2 μm (C) Localization of endogenously BAP-tagged GiMlf and V5-tagged Hsp40 at the basal bodies in trophozoites using expansion microscopy (ExM), scale bar: 10 μm. The cells were stained with anti-BAP antibody (cyan) and anti-V5 antibody (magenta), and NHS ester (primary amines of proteins, yellow). (D) Localization of endogenously BAP-tagged GiMlf and V5-tagged P26s4 at mitosomes in trophozoites of G . intestinalis using confocal microscopy. The cells were stained with anti-BAP antibody (cyan) and anti-V5 antibody (magenta), and an anti-GL50803_9296 antibody (yellow, mitosomal marker), scale bar: 2 μm The enlarged image shows a single layer of the image stack. Pearson’s correlation coefficient (ρ = 0.8448) was calculated for the subsection of GiMlf and P26s4 that colocalize in proximity to mitosomes. (E) Localization of endogenously BAP-tagged GiMlf and V5-tagged P26s4 in encysting cells of G . intestinalis in the early (5 hpi) and late (48 hpi) phases of encystation using confocal microscopy. The cells were stained with anti-BAP antibody (cyan) and anti-V5 antibody (magenta), and anti-CWP1 antibody (yellow), scale bar: 2 μm. The enlarged images show a single layer of the image stack. Pearson’s correlation coefficient (ρ) was calculated for each stack. Top panel—ρ = 0.802, middle panel—ρ = 0.742, bottom panel—ρ = 0.630.

Article Snippet: For the immunolabeling, commercial primary antibodies mouse anti-BAP (1:500, GenScript), rabbit anti-V5 (1:500, Abcam), mouse anti-acetylated tubulin (1:1000, Sigma-Aldrich–T7451), self-made primary antibodies rabbit anti-Tom40 (1:1000, [ ], rat anti-PDI2 (1:1000, [ ], rat anti-GiMlf (1:500, [ ] and commercial secondary anti-rabbit/mouse Alexa Fluor 647, anti-rat/mouse Alexa Fluor 488, and anti-rabbit Alexa Fluor 594 (1:500, Invitrogen) antibodies were used.

Techniques: Isolation, Confocal Microscopy, Staining, Microscopy, Marker

Journal: Biomedicines

Article Title: Treatment of Critical-Size Femoral Bone Defects with Chitosan Scaffolds Produced by a Novel Process from Textile Engineering

doi: 10.3390/biomedicines9081015

Figure Lengend Snippet: In vitro measurement of ( A ) cell number and ( B ) alkaline phosphatase (ALP) activity after 14 days. Cells of a human mesenchymal stromal cell line (hTERT-MSC) were seeded on the different scaffolds and cultured for 14 days in osteogenic medium to compare cell proliferation and osteogenic differentiation (mean ± SD, n = 3; * p < 0.05, ** p < 0.01; CHS: chitosan, MCM: mineralized collagen, CBA: human cancellous bone allograft).

Article Snippet: Osteoblasts were quantified by immunohistochemical staining for bone alkaline phosphatase (BAP; rabbit anti-BAP, 1:100, LINARIS Biologische Produkte GmbH, Dossenheim, Germany) and appeared as brown-stained cuboidal mononuclear cells when using 3,3′-Diaminobenzidin (DAB) as chromogen [ ].

Techniques: In Vitro, Activity Assay, Cell Culture

Journal: Biomedicines

Article Title: Treatment of Critical-Size Femoral Bone Defects with Chitosan Scaffolds Produced by a Novel Process from Textile Engineering

doi: 10.3390/biomedicines9081015

Figure Lengend Snippet: Immunohistochemical staining of osteoclasts. ( A ) Representative histological sections of tartrate-resistant acid phosphatase-positive osteoclasts (arrows) of each group and ( B ) quantitative analysis by counting the osteoclasts present in the defect area (mean ± SD, CHS: n = 9, MCM: n = 10, CBA: n = 11).

Article Snippet: Osteoblasts were quantified by immunohistochemical staining for bone alkaline phosphatase (BAP; rabbit anti-BAP, 1:100, LINARIS Biologische Produkte GmbH, Dossenheim, Germany) and appeared as brown-stained cuboidal mononuclear cells when using 3,3′-Diaminobenzidin (DAB) as chromogen [ ].

Techniques: Immunohistochemical staining, Staining

Journal: Biomedicines

Article Title: Treatment of Critical-Size Femoral Bone Defects with Chitosan Scaffolds Produced by a Novel Process from Textile Engineering

doi: 10.3390/biomedicines9081015

Figure Lengend Snippet: Immunohistochemical staining of osteoblasts. ( A ) Representative histological sections of alkaline phosphatase-positive osteoblasts (arrows) of each group and ( B ) quantitative analysis by counting the osteoblasts present in the defect area (mean ± SD, CHS: n = 9, MCM: n = 10, CBA: n = 11; * p < 0.05).

Article Snippet: Osteoblasts were quantified by immunohistochemical staining for bone alkaline phosphatase (BAP; rabbit anti-BAP, 1:100, LINARIS Biologische Produkte GmbH, Dossenheim, Germany) and appeared as brown-stained cuboidal mononuclear cells when using 3,3′-Diaminobenzidin (DAB) as chromogen [ ].

Techniques: Immunohistochemical staining, Staining